Abstract: In this research, pmaxGFP-gcc plasmid was transfected into buffalo spermatogonial stem cells cultured in vitro by Lonza nucleofector. The results showed that the highest survival rate and transfection rate of buffalo spermatogonial stem cells were obtained after comparing the transfection results of seven groups of electrical pulse optimization procedure and the corresponding optimal electrotransfection procedure was determined to be EH100,a rate of 19.1% of GFP+cells were screened by flow cytometry after 48 hours of that electrotransfection and the survival rate of transfected cells reached 74.2%. The procedure of this experiment is simple, high transfection efficiency and good repeatability. This method can be used to transfect stem cells which are difficult to transfect by common methods, and provides the basis and reference for the transgenic research of spermatogonial stem cells from other species.