In this paper, thermo-economic optimization and comparative analysis of a cascade refrigeration system configured with flash gas removal in its high-temperature cycle (HTC) and flash intercooling with indirect subcooling in lower temperature cycle (LTC) using different natural refrigerant pairs is performed. Thermo-economic optimization is carried out to maximize the exergetic efficiency and minimize the overall cost rate. The optimization model involves six design variables which include subcooling and de-superheating parameters, LTC evaporation and condensation temperatures, HTC condenser temperature and cascade temperature difference. The comparative analysis of twenty-two natural refrigerant pairs based on the results of thermodynamic and economic optimizations reveals that R717-R290 is most efficient pair and R290-R1150 is least efficient refrigerant pair thermodynamically whereas R717-R1270 is the best and R600a-R290 is the worst pair economically. Seven potential refrigerant pairs are chosen via the thermodynamic and economic optimization results and they are further compared based on their performances obtained through multi-objective optimization (maximization of exergetic efficiency and minimization of total cost rate). Multi-objective genetic algorithm is used for optimization which results in seventy non-dominated Pareto optimal solutions where the TOPSIS method is used to select a unique solution for each refrigerant pair. A comparison of refrigerant pairs using these unique solutions shows that R717-R1270 is the best refrigerant pair for the cascade system under consideration. It is also found that R717-R1270 results in 7.77% rise in COP and 5.32% reduction in overall cost when compared with NH3–CO2 refrigerant pair working under identical operating conditions.