The 30-year-old took 0.3 seconds off her own previous best of 33.758, set in New Delhi four years ago, to cross the line at the Sir Chris Hoy Velodrome in 33.435.
Compatriot Stephanie Morton, who is Meares’ room-mate, claimed silver with a time of 34.079.
Australia’s gold medalist Anna Meares (R) and Australia’s silver medalist Stephanie Morton pose on the podium after winning the women’s 500m time trial final
England’s Jess Varnish claimed the bronze in a field of 13 riders.
Four-time world champion Meares has dominated the discipline for a decade and won the 2004 Olympic gold medal in the last year the event was held before being dropped to make room for BMX.
She has now won a record-equalling five gold medals in track cycling at the Commonwealth Games, joining compatriot Bradley McGee.
It is a third successive victory in the event for Meares, who has now equalled Kathy Watt’s Australia women’s cycling record of seven Commonwealth Games medals and has become the first rider to win a medal at four Games.
Meares has the chance to beat Watt’s record on Friday when she will compete in the women’s sprint final.
If she can claim a medal in that event she will equal New Zealand’s Gary Anderson as the most decorated track cyclist at the Commonwealth Games.