Accurate isolation of targeted extracellular vesicle (EV) is challenging due to the heterogeneity of EV subpopulations which are from different cell origins. Most EV subpopulations lack a single marker whose expression cleanly distinguish them from mixed populations of closely related EVs. Herein, we developed a modular platform capable of taking multiple binding events as input, logic computations, and producing two independent outputs for tandem microchips for EV subpopulation isolation. Taking advantages of the excellent selectivity of dual-aptamer recognition-mediated DNA computation and the sensitivity of tandem microchips, this method achieves, for the first time, sequential isolation of tumour PD-L1 EVs and non-tumour PD-L1 EVs. As a result, the developed platform can not only effectively distinguish cancer patients from healthy donors, but also provides new clues for assessing immune heterogeneity. Moreover, the captured EVs can be released through a DNA hydrolysis reaction with high efficiency, which is compatible with downstream mass spectrometry for EV proteome profiling. Overall, this strategy is expected to isolate different EV subpopulations, translate EVs into reliable clinical biomarkers, and accurately investigate the biological functions of different EV subsets.