Volatile organic compounds (VOCs) of antagonistic yeasts are considered as environmental safe fumigants to promote the resistance and quality of strawberry (
Fragari
a
ananassa
). By GC‐MS assays, VOCs of
Hanseniaspora uvarum
(
H. uvarum
) fumigated strawberry fruit showed increased contents of methyl caproate (5.8%), methyl octanoate (5.1%), and methyl caprylate (10.9%) in postharvest cold storage. Possible mechanisms of
H. uvarum
VOCs involved in regulations of the defense‐related enzymes and substances in strawberry were investigated during postharvest storage in low temperature and high humidity (2 ± 1°C, RH 90%–95%). Defense‐related enzymes assays indicated
H. uvarum
VOCs stimulated the accumulation of CAT, SOD, POD, APX, PPO, and PAL and inhibited biosynthesis of MDA in strawberry fruit under storage condition. Moreover, the expression levels of related key enzyme genes, such as
CAT
,
SOD
,
APX42
,
PPO
, and
PAL6
, were consistently increased in strawberry fruit after
H. uvarum
VOCs fumigation.