Tumor-derived extracellular vesicle (EV) microRNAs (miRNAs) are important biomarkers for clinical diagnosis and disease treatment monitoring. However, the need to lyse EVs makes most methods for detection of tumor-derived EV miRNA expensive, labour-intensive, and time-consuming. Inspired by natural vesicular transport, we here developed a general strategy for in situ detection of tumor-derived EV miRNAs using aptamer-mediated selective fusion (Apt-Fusion). Taking advantages of the high selectivity, rapid and general applicability of Apt-Fusion, this method exhibits significant sensitivity and selectivity for tumor-derived EV miRNAs in a lysis-free manner using conventional flow cytometry. Using this method, the level of miR-21 in PD-L1 positive EVs was quantified and found to effectively distinguish cancer patients from healthy volunteers; additionally, miR-21 in PD-L1 positive EVs was found for the first time to correlate with tumor burden. Overall, Apt-Fusion holds great potential to detect tumor-derived EV miRNAs and expand detection of EV multi-molecular compositions, offering a new avenue for cancer diagnosis and immunotherapy response monitoring.