Optogenetically engineered cell population obtained by heterogeneous gene expression plays a vital role in life science, medicine, and biohybrid robotics, and purification and characterization are essential to enhance its application performance. However, the existing cell purification methods suffer from complex sample preparation or inevitable damage and pollution. The efficient and nondestructive label-free purification and characterization of the optogenetically engineered cells, HEK293-ChR2 cells, is provided here using an optically-induced dielectrophoresis (ODEP)-based approach. The distinctive crossover frequencies of the engineered cells and the unmodified cells enable effective separation due to the opposite DEP forces on them. The ODEP-based approach can greatly improve the purity of the separated cell population and especially, the ratio of the engineered cells in the separated cell population can be enhanced by 275% at a low transfection rate. The size and the membrane capacitance of the separated cell population decreases and increases, respectively, as the ratio of the engineered cells grows in the cell population, indicating that successful expression of ChR2 in a single HEK293 cell makes its size and membrane capacitance smaller and larger, respectively. The results of biohybrid imaging with the optogenetically engineered cells demonstrated that cell purification can improve the imaging quality. This work proves that the separation and purification of engineered cells are of great significance for their application in practice.