(E)-2-hexenal fumigation inhibits the growth of Botrytis. cinerea, but the direct target and its effect on postharvest strawberry have not yet been discovered. In the present study, we applied increasing level of (E)-2-hexenal fumigation from 0.524 μM to 1048 μM to B. cinerea on strawberry and medium. Results showed that (E)-2-hexenal fumigation inhibited lesion diameter on strawberry by 13.96 %, 20.41 % and 100 % with the dosage increasing. On medium, (E)-2-hexenal fumigation increased both the ROS and mitochondria membrane potential level of B. cinerea. LC-MS/MS and FTIR results demonstrate a 1:1 Michael addition reaction between (E)-2-hexenal and glutathione with the product GSH-H in B. cinerea under (E)-2-hexenal fumigation. Furthermore, the consumption of glutathione and glutathione disulfide along with the production of GSH-H during fumigation in B. cinerea caused by (E)-2-hexenal were both concentration- and time-dependent. This study locates the direct target and discovered the functional model of (E)-2-hexenal to B. cinerea.