This chapter presents the operational principles of the novel ejector-enhanced auto-cascade refrigeration (NEACR) cycle, which is suggested as an alternative to conventional cascade refrigeration cycles. It is particularly designed for applications requiring low and ultra-low temperatures. The main purpose is to evaluate the NEACR cycle performance under various operating conditions, taking into account parameters such as volumetric cooling effect, total compression work rate, coefficient of performance (COP), total exergy destruction rate, exergy efficiency of the entire system, and sustainability index. Energy and exergy analyses are employed to assess the NEACR cycle using the R170/R600 refrigerant mixture at different evaporation and condensation temperatures. The findings reveal that the COP reaches its peak value of 0.70, and the exergy efficiency reaches 26.5% when the condensing temperature is reduced to 20 °C, with a mass fraction of 0.48/0.52 for R170/R600 refrigerant, achieving an evaporation temperature of −60 °C. Lowering the condensing temperature from 40 °C to 20 °C leads to a significant improvement, with COP and exergy efficiency increasing by 30.9%, and the sustainability index improving by 42.0%. Additionally, there is a decrease in total compression work rate by 23.6% and total exergy destruction rate by 29.6%.