The detection and separation of biological samples are of great significance for achieving accurate diagnoses and state assessments. Currently, the detection and separation of cells mostly adopt labeling methods, which will undoubtedly affect the original physiological state and functions of cells. Therefore, in this study, a label-free cell detection method based on microfluidic chips is proposed. By measuring the scattering of cells to identify cells and then using optical tweezers to separate the target cells, the whole process without any labeling and physical contact could realize automatic cell identification and separation. Different concentrations of 15 µm polystyrene microspheres and yeast mixed solution are used as samples for detection and separation. The detection accuracy is over 90%, and the separation accuracy is over 73%.