Circulating tumor cell (CTC) analysis has been approved for cancer diagnosis and monitoring. However, efficient sorting and high-through phenotypic counting of CTCs from peripheral blood is still a challenge. In this manuscript, we propose an optofluidic flow cytometer (OFCM), which integrates a multistage microfluidic chip and a four-color fluorescence detection system. The OFCM can automatically complete CTC separation, 3D focusing in the microchannel, single-cell phenotypic analysis, and counting at 1.2 mL of whole blood/hour. A high recovery greater than 95% was obtained. Using the OFCM, we analyzed the epithelial-to-mesenchymal transition (EMT) phenotype of CTCs in patients with breast cancer and patients with nonsmall cell lung cancer, which proved that the OFCM is adaptable for phenotypic counting of various CTCs based on the fluorescence labeling of varied biomarkers. We believe that this OFCM will provide a convenient and efficient device for clinical liquid biopsy of tumors.