The 40-year-old woman was working on a tour bus with sightseers from Wuhan, the epicentre of the outbreak, in January.
She was first confirmed as infected with the coronavirus on January 29.
After being discharged from hospital she tested negative for the virus on February 6, although she still had a cough at the time.
She had no symptoms a week later, but returned to the doctor on February 21 complaining of a sore throat and chest pains.
On Wednesday, she tested positive for the coronavirus for a second time, officials in Osaka said.
The driver of the tour bus also tested positive for the virus.
At least 186 people in Japan have so far contracted the virus, with three deaths in the country linked to the outbreak.
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