Tumor-derived exosomes have been recognized as potential biomarkers for cancer diagnosis because they are actively involved in cancer progression and metastasis. However, progress in practical exosome analysis is still slow due to the limitation in exosome isolation and detection. The development of microfluidic devices has provided a promising analytical platform compared with traditional methods. In this study, we develop an exosome isolation and detection method based on a microfluidic device (ExoDEP-chip), which realized microsphere mediated dielectrophoretic isolation and immunoaffinity detection. Exosomes were firstly isolated by binding to antibodies pre-immobilized on the polystyrene (PS) microsphere surface and were further detected using fluorescently labeled antibodies by fluorescence microscopy. Single microspheres were then trapped into single microwells under the DEP force in the ExoDEP-chip. A wide range from 1.4 × 103 to 1.4 × 108 exosomes per mL with a detection limit of 193 exosomes per mL was obtained. Through monitoring five proteins (CD81, CEA, EpCAM, CD147, and AFP) of exosomes from three different cell lines (A549, HEK293, and HepG2), a significant difference in marker expression levels was observed in different cell lines. Therefore, this method has good prospects in exosome-based tumor marker detection and cancer diagnosis.