According to the "Montreal Protocol", the R22 used in the refrigeration system of fishing vessels needs to be banned before 2030 due to its effect on the depletion of the ozone layer. Therefore, it is imminent to find an ideal alternative refrigerant. By screening and proportioning the components of the mixed refrigerants, it can not only meet the refrigeration requirements, but also meet the environmental requirements, and become the mainstream trend to replace traditional refrigerants. In this paper, a single-stage self-cascading refrigeration system is designed, and the chemical process simulation software is used to carry out the application substitution research of mixed refrigerants. Based on the characteristics of the self-cascading refrigeration system and the thermophysical properties of the mixed refrigerants, R14, R23, R134a, and R245fa, were selected for the target temperature zone of -65 ℃, which were composed of binary, ternary, and quaternary mixed refrigerants respectively. For the self-cascading refrigeration system, the thermodynamic calculation and analysis of binary, ternary and quaternary mixed refrigerants under different components and different concentrations were carried out. The results show that the refrigeration system with quaternary mixed refrigerants is better than that with binary and ternary mixed refrigerants. When the ratio of quaternary refrigerant components is R14/R23/R134a/R245fa (0.35/0.36/0.1/0.19), the COP of the refrigeration system could reach 1.037, the exhaust pressure and the suction pressure are 1590 kPa and 740 kPa respectively, the pressure ratio is less than 3, and the exhaust temperature is only 61℃. Compared with the traditional R22 refrigeration system, the COP of the mixed refrigerant system is close to the traditional R22 refrigeration system. In terms of volume refrigeration efficiency, the suction flow rate is only 26.56% of the first-stage suction flow rate of the R22 refrigeration system, which can reduce the size of the compressor in the system, making the system simpler and more cost-effective. Therefore, the new mixed refrigerants of R14/R23/R134a/R245fa have the potential to replace the traditional refrigerants of R22.