Tumor-derived exosomes have been recognized as promising biomarkers for early-stage cancer diagnosis, tumor prognosis monitoring and individual medical treatment. However, it is a huge challenge to separate exosomes from trace biological samples in clinics for disease diagnosis. Herein, we propose a simple, quick, and label-free method for isolating circulating exosomes from serum of patients. The strategy synergistically integrates chitosan electrostatic-adsorption, micro-patterned substrates, and microfluidic shuttle flow control to enable the capture/release of circulating exosomes in a simple manner. Using this microchip, we can isolate exosomes from trace samples (10 μl) with relative purity over 90% and high RNA recovery ratio over 84% within 15 minutes, which is impossible for traditional ultracentrifugation methods. We then validate the application of the microchip using 24 serum samples from clinical breast cancer and breast fibroma patients. The isolated exosomes are subjected to miRNA sequencing and RT-PCR, followed by pathway prediction analysis. The results showed that exosomes were relevant to the invasion and metastasis of breast cancer cells and hsa-miR-18a-3p might have the potential to become a new biomarker for distinguishing breast cancer from breast fibroma (AUC = 0.83, P value = 0.019). This established method is simple, quick and easy to operate with integration. And it may pave a new way for clinical research on exosomes and tumor relevant diagnosis.