Ambrose, who sensationally stepped aside from his lead driver role at DJR Team Penske earlier this season, finished behind the Fords of Mark Winterbottom and Chaz Mostert.
After making his return to V8 Supercars this year following almost a decade driving NASCAR, Ambrose was competing for the first time since the Formula 1 Australian Grand Prix in March.
"These endurance races are going to be a lot of fun for me. I'm enjoying my time in the car, Scott Pye's been a great team mate and I'm really looking forward to seeing how we go," he said.
"The cars have changed a lot. My head is used to going 200 miles an hour and not slowing down too much, and the feel from those cars in NASCAR is very different to these (V8 Supercars) so it's just taking a bit of unwinding."
Jamie Whincup and co-driver Paul Dumbrell topped the overall timesheets for the day, with the pair looking good to take an historic Sandown 500 three-peat this weekend in a brand new car.
"It was a risk, but we've got to take risks at this stage, not being at the pointy end of the championship, as we would like. The risk paid off this time and the car is good. It's just a matter of making it quick this time," said Whincup.
A tenth of a second behind the Red Bull Racing Australia pair were young guns Chaz Mostert and Cameron Waters. Waters showed blistering speed in both co-driver sessions, finishing first and second in each, respectively. Mostert earlier took the #6 Ford to second place behind Whincup.
"We rolled out pretty strong today. The car was making sense, as it has been at most of the rounds this year. We'll give it a fine tune and see if we can make it a little bit quicker," said Mostert.
There is one final practice session tomorrow morning, before two qualifying races to determine the grid for Sunday's race.
MARCOS AMBROSE AUDIO:
Marcos Ambrose says the endurance races will be fun for him, and he is enjoying driving the car.