In an expanding global energy market, an efficiency improvement of cooling systems plays an important role to achieve sustainability goals. A large part of energy efficiency studies involve system solutions that use natural refrigerants, such as the carbon dioxide, which are popular due to their environmental friendliness and energy efficient when compared to synthetic fluids. However, a CO2 based system obtains significant COP degradation at high ambient temperature, which forces to apply novel solutions. One of the promising technologies which covers system improvements with natural refrigerants is an ejector-aided cycle. The ejector allows a COP improvement over 20% in cooling systems, especially under high ambient temperature conditions. Nevertheless, the complexity of an ejector design and a control solution of the ejector-based system required further development in terms of the COP improvement and the control strategy. One of the idea is to introduce a thermoelectric sub-cooling method into the CO2 ejector-based system. Thermoelectric subcooling method uses thermoelectric modules on heat exchanger, by using the Peltier Effect in thermoelectric modules it helps to reduce temperature in the heat exchanger and helps system improvement with subcooling. Hence, the main aim of this work is to perform numerical and experimental research of the proposed thermoelectric sub-cooler applied into the CO2 refrigeration system at cooling capacity of 50 kW. The thermoelectric sub-cooler device designed for 1.8 kW nominal cooling capacity. Additionally, numerical and experimental work evaluated. Numerical investigation conducted to investigate influence of thermoelectric modules operations to temperature distribution inside of the sub-cooler unit meanwhile the experimental investigation conducted to investigate impact of the sub-cooler unit operation on different CO2 operating conditions.