The invention discloses a disinfection method of a grape explant in tissue culture. The disinfection method comprises the following steps: a grape stem section is washed with sterile water for 3-5 times and then washed with 75% alcohol for 30-50 seconds, and a grape stem section after alcohol washing is obtained; the grape stem section is washed with the sterile water for 3-5 times and soaked in 800-1,000 times of the 84 disinfectant for 2-4 minutes, and the grape stem section after 84 disinfectant soaking is obtained; the grape stem section is washed with the sterile water for 3-5 times and soaked in 0.1% mercuric chloride for 5-8 minutes, and the grape stem section after mercuric chloride soaking is obtained; the grape stem section is washed with the sterile water for 3-5 times, and disinfection is finished for inoculation. The disinfection method has the following benefits: after the disinfection method of the grape explant in tissue culture is adopted for disinfection, the explant is inoculated to a medium for culture, the mould generation rate after rooting and leafing can be reduced by more than 60%, and accordingly, the method can effectively reduce mould contamination of the explant after inoculation.