Dimethyl sulfoxide (Me₂SO) in cell banking exhibits significant side effects on both the cells and the human body. Therefore, an approach that mitigates the side effects of Me₂SO with comparable efficacy is urgently needed. The human umbilical cord mesenchymal was used as the research material. First, the thermal physical properties of trehalose, glucose, and L-proline and their regulation of ice crystal growth were measured using a differential scanning calorimeter and a cryomicroscope. Cryopreservation experiments were performed to determine the optimal concentration of each component in the cryopreservation solution, and the viability and functionality of the cells after cryopreservation were validated. The results show that there is no significant difference in cell viability (92.42%±0.28%) and recovery rate (87.80%±4.22%) between the use of the novel stem cell cryopreservation solution (1.25 mol/L ethylene glycol+10 g/L whey protein+0.1 mol/L trehalose+Normosol-R) and the conventional cryopreservation solution (a volume fraction of 10% Me₂SO). Moreover, after 3 days of culture, the cell number was (12.42±0.60) × 10⁶ (proliferation fold of 4.97), and the cell phenotype was not significantly different from that of fresh cells. The proposed novel solution for stem cell cryopreservation solves the problem of "Me₂SO-free" cryopreservation of cells and offers promising potential for clinical applications.