The invention discloses a culture method of oral squamous cell carcinoma organoid. The culture method comprises the following steps: S1, preparing a dissociation enzyme; S2, preparing a culture medium, wherein the culture medium is composed of a basal culture medium DMEM/F12, an R-spondin1 conditional culture medium, a Wnt3A conditional culture medium, sterile water and functional components; S3, obtaining a sample through clinical operation, namely obtaining a tumor specimen through material taking or surgical resection, cutting tissue, washing the tissue with sterile normal saline for three times, soaking the tissue in a DMEM medium for 1 hour and continuing to dissociate; S4, tissue pretreatment, namely placing the sample obtained in the step S3 in a sterile 6-well plate, and cutting up sample tissue blocks by using a disposable sterile surgical blade; S5, tissue dissociation and digestion, namely adding the dissociation enzyme prepared in the step S1, repeating the process of centrifugation-supernatant removal for three times so as to fully remove the dissociation enzyme, and mixing the cells with the sample tissue blocks before the last time of centrifugation; and S6, carrying out organoid culture.