The 2012 winner outsprinted Ruth Corset, who won in 2010, at the end of the 102km road race over a testing circuit at Buninyong, near Ballarat.
Spratt's Orica-AIS teammate Rachel Neylan finished a few metres behind her and Corset for the bronze medal.
"The first one was probably a little bit more unexpected, this time I came in knowing that my form was really good," Spratt said.
"Having Sarah Roy out there in that early break was great for us. It was a great situation and it meant we could not panic, sit in there and relax and certainly had the other teams wondering what we were going to do. Once we started making those moves it really meant business.
"Getting across to Sarah was great, she was cramping, she was absolutely done but she just said, "What can I do, What do I do?" she just gave me every last bit of energy she had and I knew that it was up to me to finish it off."
The race got cracking inside the final 25km when Corset and Spratt attacked heading into the penultimate lap.
As the chasers crossed the finish line with two laps remaining the advantage of early breakaways Sarah Roy and Louisa Lobigss was down to one minute and 10 seconds on the two chasers, with former criterium champion Kimberley Wells bridging across, and under two minutes to the peloton.
Roy and Lobigs's time out front under the hot sun started to take its toll, with Spratt, Corset and Wells joining them in the lead. Starting the final lap the five riders held a touch and go advantage of 42 seconds on a much-reduced peloton.
On reaching the final climb Spratt and Corset took the opportunity to distance the faster finishers and continue on solo. With the remaining riders caught a flurry of attacks set off from the peloton with three former road race medallists Katrin Garfoot, Rachel Neylan and Lauren Kitchen taking one last opportunity to go across.
With Spratt having teammates in the move behind, Corset was forced to do much of the work at the front with Spratt reluctant to contribute as the chasers closed in.
Entering the final kilometre the visibly closing gap was down to just 16 seconds with the three frantically chasing to make it across.
Coming from second wheel Spratt opened her sprint, taking an immediate advantage and holding it to the line to claim gold.
Run at the same time as the elite women, the under 23 road race was won by Jenelle Crooks (QLD) ahead of Jessica Mundy (SA) with Ellen Skerritt (QLD) third.