Vondrousova, who won her lone Grand Slam title at Wimbledon in 2023, has struggled for form since a quarter-final run at the US Open last year, making several first-round exits.
"I'm really sorry that I had to withdraw from the Australian Open due to ongoing shoulder issues," she wrote on Instagram.
"After everything I've been dealing with, I need to prioritise my health, even though this decision wasn't easy. Thank you all for your understanding and support."
Vondrousova was the 32nd seed and was scheduled to play American Hailey Baptiste in the first round.
Former Wimbledon finalist Matteo Berrettini was also forced to withdraw from the year's first major due to discomfort in his abdomen.
"I won't be ready to perform at my best level for five sets. It hurts because playing in Australia has always been special. After four weeks of intense training I was excited and ready to get going," he said.
Berrettini was set to play sixth seed and local favourite Alex De Minaur, who will now face American Mackenzie McDonald.