Bladder cancer (BC) often requires lifetime monitoring due to its high recurrence rate. Exfoliated bladder cancer cells (EBCCs) may express a series of different biomarkers according to its epithelial-mesenchymal transition (EMT) status, a phenomenon characterized by loss of intercellular adhesion, enhanced cell motility, and cancer invasion. Here, we demonstrated the clinical heterogeneity of EBCCs using an integrated microfluidic assay to separate various EMT subtypes of EBCCs in real-time and under high-throughput based on the principle of inertial focusing. Enriched cells from BC patient-derived urine bladder wash samples were isolated based on cell size and characterized by antibodies targeting EMT biomarkers such as cytokeratin (CK), vimentin (VIM), survivin, and epidermal growth factor receptor (EGFR). This rapid, non-invasive method demonstrates high efficiency of cancer cell recovery under the optimal flow rate and the specific retrieval of various EMT phenotype cell fractions from respective device outlets. The evaluation of clinical samples revealed a vast amount of tumor heterogeneity, reflecting different EMT phenotypes, which can correlate with drug resistance and tumor dormancy. Overall, the separation of heterogeneous clinical samples can better facilitate routine screening procedures and greatly enhance personalized treatment.