The Stage 3 victory gives Spratt a commanding 29sec overall lead on Lauren Stephens (Cylance) and previous race leader Katrin Garfoot (UniSA Australia) with one day of action left.
Grace Brown (Holden Team Gusto) finished third on the stage, behind Spratt and Stephens.
Linda Villumsen (Team Virtu Cycling) launched the first attack which netted her a two minute lead on the peloton.
Spratt and Stephens then set off in pursuit and quickly caught and dropped the former world time trial champion.
The pair worked well together until the climb at Comet Mine, where Spratt kicked into gear on the steep 12.2 per cent gradient to grab the mountain points.
Spratt then dug deep dig deep in the final two kilometres to the finish, collapsing from the effort after she crossed the line.
“The team were just amazing," Spratt said. "Linda Villumsen got out there and got that big gap and we didn’t panic, actually Annemiek (Van Vleuten) was there and she really calmed me down because I was stressing.
“UniSA started to panic and they started to send riders up the road and we actually got in an ideal move with Annemiek and Lucy (Kennedy) in there with Kat (Garfoot) behind.
"Kat panicked and had to do some work. I sat on her and as soon as she caught the group, I attacked straight away.”
“I knew it was the perfect moment. We did exactly what we wanted, we isolated Kat, and I could capitalise off that.”
The team effort was rewarded with Spratt also claiming the mountains and sprint classifications.
The race ends on Sunday with the Adelaide street race and Spratt's powerful Mitchelton-Scott team will have little trouble protecting her lead.
Amanda Spratt is on the verge of successfully defending her Santos Women's Tour Down Under title after gutsy Stage 3 win at the Adelaide Hills town of Hahndorf. pic.twitter.com/TQ673RhvMY
— CyclingCentral (@CyclingCentral) January 13, 2018