Saccharomyces cerevisiae (S. cerevisiae) has been classically used to treat diarrhea and diarrhea-related diseases. However, in the past two decades, fungal infections caused by S. cerevisiae have been increasing among immunocompromised patients, and it takes too long to isolate S. cerevisiae from blood to diagnose it in time. In this paper, a new method for the isolation and selection of S. cerevisiae from red blood cells (RBC) is proposed by designing a microfluidic chip with an optically-induced dielectrophoresis (ODEP) system. It was verified by theory and experiments that the magnitude and direction of the dielectrophoresis force applied on RBCs and S. cerevisiae are different, which determine that the S. cerevisiae can be isolated from RBCs by the ODEP system. By designing the specific light images and the dynamic separation mode, the optimal operating conditions were experimentally achieved for acquiring higher purity of S. cerevisiae. The purity ranges were up to 95.9%-97.3%. This work demonstrates a promising tool for efficient and effective purification of S. cerevisiae from RBCs and provides a novel method of S. cerevisiae isolation for the timely diagnosis of fungal infections.