Circulating extracellular vesicles (EVs) have emerged as a valuable source of cancer biomarkers. However, the high degree of EV heterogeneity and the complexity of clinical samples pose a challenge in the sensitive identification of tumor‐derived EVs. Here we introduce a one‐step thermophoretic AND gate operation (Tango) assay that integrates polyethylene glycol (PEG)‐enhanced thermophoretic accumulation of EVs and simultaneous AND gate operation on EV membranes by dual‐aptamers recognition. By using the Tango assay to detect tumor‐derived EVs with co‐presence of EpCAM and PSMA directly from serum in a homogeneous, separation‐free format, we can discriminate prostate cancer (PCa) patients from benign prostatic hyperplasia (BPH) patients in the diagnostic gray zone with an accuracy of 91 % in 15 min. Our approach streamlines EV enrichment and AND gate operation on EVs in a single assay, providing a rapid, straightforward, and powerful method for precise and non‐invasive diagnosis of cancer.