Cryopreservation of spermatogonial stem cells (SSCs) is a necessity to preserve the genetic information of valuable livestock herds and to produce transgenic animals. However, serum, a key component that allows efficient cryopreservation, has many limitations attributed to its undefined composition, inter-batch variations, and contamination potential. Therefore, we aimed to establish a method for serum-free cryopreservation of SSCs. To evaluate the cryopreservation efficiency of serum replacements, we assessed the recovery rate, relative proliferation potential, proliferation capacity, and apoptosis capacity. SSCs were characterized, and their functional activity was determined through immunofluorescence, RT-qPCR, and spermatogonial transplantation. The efficiency of each serum replacement was compared to that of the negative control (10% DMSO in DPBS) and positive control (10% DMSO and 40% FBS in DPBS). Our results indicated that cryopreservation with 5% human serum albumin (rHSA) exhibited a higher relative proliferation potential (274.0 ± 13.4%) than with DMSO control (100 ± 8.6%), with no significant difference from the 40% FBS (190.0 ± 20.1%). Moreover, early apoptosis also significantly decreased to a greater extent with 5% rHSA (5.1 ± 0.7%) than with DMSO control (12.9 ± 0.8%) and was at a level comparable to the 40% FBS (4.9 ± 0.8%). In addition, the SSCs cryopreserved with 5% rHSA exhibited normal self-renewal and differentiation abilities. In conclusion, 5% rHSA is a potential serum replacement for SSC cryopreservation, with properties comparable to that of serum. These results would contribute to the application of SSCs in improving livestock and in future clinical trials for human infertility treatment.