In order to explore the feasibility of using binary zeotropic mixtures to replace pure fluids, a subcritical organic Rankine cycle thermodynamic model based on mixtures was established. A screening method of mixtures was proposed on the basis of the boiling point difference. The net power output was selected as the optimization target for the evaporation parameter and mass ratio. For different heat source temperatures, the best mixtures were selected. The system performance parameters and energy loss distribution of the best mixtures and pure fluids are compared. The results show that the net power output of the best mixture is greater than that of the best pure fluid at the same heat source temperature with an increment ranging from 0.13% to 5.04%, which mainly results from mixtures’ smaller latent heat of vaporization and cooling temperature glide closing to cooling water temperature rise. The inlet temperature and pressure of the expander with mixtures are lower than those of pure fluids, and the decrement can reach up to 9.93% and 27.08% respectively. Compared with the best pure fluids, the best mixtures have lower exergy loss in the expander and condenser, which makes the total exergy loss of mixtures lower than that of pure fluids.