The present disclosure relates to a dissociation reagent for tumor tissues. The dissociation reagent does not contain collagenase or trypsin but further contains hyaluronidase or a mixture of hyaluronidase and DNase I. The present disclosure also relates to use of the dissociation reagent in dispersing tumor tissues and detecting expression level of molecular markers on cell surface by flow cytometry. The dissociation reagent of the present disclosure does not cause degradation of molecular markers on cell surface such as CD8, PD-1, Tim-3, Lag-3 and the like, thus does not affect downstream assays.