This paper presents results of a preliminary experimental study of the Organic Rankine Cycle (ORC) using CO2/R134a mixture based on an expansion valve. The goal of the research was to examine the feasibility and effectiveness of using CO2 mixtures to improve system performance and expand the range of condensation temperature for ORC system. The mixture of CO2/R134a (0.6/0.4) on a mass basis was selected for comparison with pure CO2 in both the preheating ORC (P-ORC) and the preheating regenerative ORC (PR-ORC). Then, the feasibility and application potential of CO2/R134a (0.6/0.4) mixture for waste heat recovery from engines was tested under ambient cooling conditions. Preliminary experimental results using an expansion valve indicate that CO2/R134a (0.6/0.4) mixture exhibits better system performance than pure CO2. For PR-ORC using CO2/R134a (0.6/0.4) mixture, assuming a turbine isentropic efficiency of 0.7, the net power output estimation, thermal efficiency and exergy efficiency reached up to 5.30 kW, 10.14% and 24.34%, respectively. For the fitting value at an expansion inlet pressure of 10 MPa, the net power output estimation, thermal efficiency and exergy efficiency using CO2/R134a (0.6/0.4) mixture achieved increases of 23.3%, 16.4% and 23.7%, respectively, versus results using pure CO2 as the working fluid. Finally, experiments showed that the ORC system using CO2/R134a (0.6/0.4) mixture is capable of operating stably under ambient cooling conditions (25.2–31.5 °C), demonstrating that CO2/R134a mixture can expand the range of condensation temperature and alleviate the low-temperature condensation issue encountered with CO2. Under the ambient cooling source, it is expected that ORC using CO2/R134a (0.6/0.4) mixture will improve the thermal efficiency of a diesel engine by 1.9%.