The event gets under way on Sunday, raising money for cancer treatment and commemorating the 73-year-old who died last year after battling breast cancer.
Thousands of walkers and hundreds of dogs are expected to gather at one of the Grease star's favourite spots, Melbourne's Alexandra Gardens.
It will be the second Walk for Wellness since the singer died in 2022 after battling breast cancer.
Walkers will cover 5km around The Tan track from 9am, with "virtual" participants also invited to join in anywhere in the world.
Money raised goes towards wellness programs at the Olivia Newton-John Cancer Wellness Centre (ONJ Centre) based at Austin Hospital in northeast Melbourne.
Sponsorship and donations will help fund the centre's evidence-based therapies that ease the side effects of cancer treatment and support people emotionally, spiritually and physically.
Dog yoga, wellness tents, craft activities and food vans will provide entertainment, and the event wraps up with a concert featuring Newton-John's niece Tottie Goldsmith and singers Phil Burton and David Campbell.
A state memorial was held for Olivia Newton-John in February at Melbourne's Hamer Hall.
Newton-John, best known for playing Sandy in the 1978 hit Grease opposite John Travolta, died on August 8 last year.
She battled breast cancer three times after first being diagnosed in 1992, leading her to create the Olivia Newton-John Foundation to fund cancer research.
Last year's Walk for Wellness was held just months after the singer's death, and kicked off with a singalong to her Grease hit Hopelessly Devoted to You before the crowd, including 200 registered dogs, got moving.
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