Ambrose began racing karts at the age of ten. He won four Tasmanian state junior karting titles and was the Australian karting champion in 1995 in the Clubman Heavy class at the Dubbo circuit in New South Wales.
He moved into Formula Ford in 1996. The Ambrose family have a rich history in this formula as Marcos' father Ross Ambrose, along with Ralph Firman Sr, co-founded Formula Ford chassis builder Van Diemen. Ambrose finished second in the Australian Formula Ford championship in 1997.
In 1998 Ambrose moved to Europe in a bid to reach Formula 1, competing in British Formula Ford in 1998 and 1999. In 1999 he won the European Formula Ford Championship. In 2000 he began the season racing in the French Formula 3 Championship, before switching mid-season to the British Formula 3 Championship.
At the end of 2000 Ambrose did not have the budget to continue in racing in Europe, and returned to Australia.. In October 2000 he was invited to compete in a Young Guns invitational race held at the Lexmark Indy 300. Ambrose won against a host of young drivers in Honda road cars.
For 2001 Ambrose was signed by Stone Brothers Racing to drive the Pirtek Racing Ford Falcon. Ambrose stunned the V8 Supercar world when he qualified on pole for his first ever V8 Supercar race, supporting the Australian Grand Prix. Ambrose went on to finish eighth in the championship, winning the Rookie of the Year award.
The 2002 season saw Ambrose start the season snaring the pole position at Phillip Island before recording his debut race win in the first race. He eventually finished third in the V8 Supercar Championship.
With a new BA Falcon, Ambrose gave the Ford team a great start to the 2003 season with victory in the first race of the Clipsal 500 in Adelaide. He followed this fantastic start with a third career win at Eastern Creek Raceway where he led Ford home to a memorable 1-2-3 at the Sydney track. This was Ford's first clean sweep of the podium since Eastern Creek 1997 and Ambrose’s first win for the season. After 13 rounds in the 2003 V8 Supercar Series, Ambrose was presented with the driver’s series trophy, 102 points clear of second place.
In 2004 Ambrose claimed three pole positions and five round wins and went into the final round at Eastern Creek Raceway with a virtually unbeatable lead. In the end he collected his second championship in the opening Saturday night race and then went on to clean-sweep the round in record-breaking style in his Pirtek Falcon. This was the first time Ford had won back-to-back titles since 1988-89. Teammate Russell Ingall also drove a superb final round to finish second in the championship, giving Stone Brothers Racing a memorable 1-2 Quinella finish.
Ambrose won the coveted Barry Sheene Medal in 2003 and 2004.
He started 2005 the way he ended 2004 with a clean sweep of the opening round in Adelaide. He remained in the championship lead until Round 10 at the Bathurst 1000 when he was involved in a controversial crash with Greg Murphy approaching BP Cutting on the way 'up The Mountain', with whom he shared some heated words after the crash. This crash, combined with a poor perfomance at the co-hosted (with the Champ Cars) round on the Gold Coast arguably lost him a third straight title. He thereafter supported teammate Russell Ingall's title bid successfully, and the two helped Ford and Stone Brothers Racing take the Drivers, Teams, and Manufacturer's championships.
He thereafter supported teammate Russell Ingall's title bid successfully, and finished third in the championship behind the aformentioned Ingall, and fellow Ford driver, Craig Lowndes. The three drivers also helped Ford to the manufacturers title.
2006 NASCAR Craftsman Truck Series
At the first V8 Supercar race of 2005, Ambrose called a press conference on the Saturday morning to announce that he would leave V8 Supercars at the end of the 2005 season to try and make a career in NASCAR.
The Ford Motor Company signed Ambrose to participate overseas in the United States with Wood Brothers/JTG Racing in the NASCAR Craftsman Truck Series in 2006. However, Ambrose had to wait until part way through the 2006 season to begin. NASCAR did not clear Ambrose to race the NCTS' first three races, as they were held on intermediate to high-speed ovals, and like fellow Wood Brothers/JTG Racing driver Bobby East, Ambrose was not cleared to start in the faster races. Ambrose made his Truck Series debut on April 1, 2006 at the Kroger 250 at Martinsville Speedway, qualifying 20th and finishing 33rd after being caught up in an incident unfolding in front of him.
Ambrose is the first notable Australian driver in a national NASCAR series event since Dick Johnson in 1990.
Ambrose made history by finishing third in the O'Reilly Auto Parts 250 at Kansas Speedway on July 2, 2006. This was the first time a non-American driver has finished in the top five of a truck series event since Canadian Ron Fellows won on the Watkins Glen road course on June 26, 1999. He also took the lead in the opening laps, becoming the first Australian to lead laps in NASCAR Craftsman Truck Series competition. The next week, he made further history by scoring his first Bud pole position for the Built Ford Tough 225 at Kentucky Speedway , and then leading the most laps in the race itself, though he finished 19th.  Ambrose scored his second podium result of his debut Craftsman Truck Series season, finishing 3rd in the Toyota Tundra 200 at Nashville Superspeedway after qualifying 11th.
Team Australia/Aussie Vineyards
Team Australia is a multi-levelled motorsport program headed by Australian businessman Craig Gore. It became the naming rights sponsors of Marcos Ambrose’s entry in the NASCAR Craftsman Truck Series in 2006 after his first race.
Team Australia are already the sponsor of two-car Champ Car and Champ Car Atlantic Championship teams in the U.S., alongside several motorsport interests in Australia. The new sponsorship will be weaved into a nation-wide marketing campaign designed to promote quality Australian and Australian-themed products and Australia as a tourism destination.