Extracellular vesicles (EVs) are cell-derived nanoscale vesicles that provide promising biomarkers for the non-invasive diagnosis of cancer because they carry important cancer-related DNA, RNA and protein biomarkers. However, the clinical application of EVs is limited by tedious and non-standardized isolation methods that require bulky instrumentation. Here, we propose an easy-to-operate, simple dielectrophoretic (DEP) method for EV isolation with higher recovery efficiency (>83%) and higher purity than ultracentrifugation (UC). The DEP chip reduces the isolation procedure from 8 h to 30 min. To facilitate subsequent analysis, our DEP chip achieved integration of EV isolation and in situ lysis of EVs for the first time. Our chip also achieved on-chip siRNA delivery to EVs isolated by DEP. We found that EVs isolated from the plasma of lung cancer patients contained higher levels of miR-21, miR-191 and miR-192 compared to those from healthy people. With on-chip detection, EGFR in EVs could distinguish lung cancer patients from healthy people. Overall, this study provides an efficient and practical approach to the isolation and detection of EVs, which could be used for the early diagnosis of lung cancer.