The woman was pronounced dead late on Tuesday at a private hospital on the island after the attack during a night-time swim at Lamai beach.
"Doctors said she died due to poison from a box jellyfish," Lieutenant Thanakorn Patnankaew, a tourist police officer on the island, said.
He added that local officials have since travelled to nearby beaches to warn tourists to be more cautious while swimming, though such stings are rare.
The German Consulate in Phuket confirmed that one of its citizens was killed due to a fatal jellyfish sting but could provide no further details.
In August a Thai woman died due to a box jellyfish sting off Koh Phangan, an island a short ferry ride away from Koh Samui that is especially popular with backpackers for its hedonistic full-moon beach parties.
And in August 2014, a five-year-old French boy was killed in an attack by the species in the same area.
The box jellyfish has trailing tentacles two to three metres (six to 10 feet) long that can pack a lethal sting for swimmers. It feeds on small fish and crustaceans.
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