Abstract: The present disclosure provides a method for separating SSCs from a frozen testicular tissue, and belongs to the field of cell biology. In the present disclosure, the method includes the following steps: resuscitating a frozen testicular tissue, digesting the resuscitated testicular tissue with a collagenase IV and a DNase I until seminiferous tubules are exposed and tube walls become coarse; washing the digested testicular tissue with a DPBS, conducting centrifugation and culture until cells migrate; collecting the migrated cells, and enriching and purifying SSCs. The method can maintain a milieu interieur of the SSCs to the greatest extent, and therefore cell viability, and less cell damages. Therefore, the method can be used for the separation of SSCs from frozen testicular tissues.