Extracellular vesicle (EV)-derived microRNA (miRNA) has been reported to be a novel biomarker for diagnosis of ovarian cancer (OvCa). This study reported an integrated microfluidic system to automate extraction and quantification of EV-miRNA on a single chip within 120 min. The EV capture rate was 1.8-fold higher than our previous work (45%) due to the use of anti-CD63 beads and anti-EpCAM beads. Moreover, two miRNA biomarkers (i.e. miR-21 and miR-200a) were ultaneously detected and quantified with multiplex PCR while the resulting calibration curves exhibited a high linearity without false-positive signals, which could be carried out on a digital PCR module. With this approach, a precise and specific diagnostic tool for OvCa could be feasible.