Noninvasive detection of small extracellular vesicles (sEVs) has become one of the most promising liquid biopsy methodologies for effective and timely cancer diagnosis and prognostic monitoring. Currently, accurate and sensitive detection of tumor-derived sEVs is compromised by their heterogeneous nature, and the tissue origin and parent cell cycle change may significantly affect the tumor-associated information (e.g., phenotypic proteins) of sEVs. Accordingly, many of the single-marker dependent detections on sEVs may not provide comprehensive information about the tumor, and their reliability and clinical applicability cannot be guaranteed. Herein, a strategy for constructing AND gate photoluminescence on tumor-derived sEVs is proposed. Briefly, only after co-recognition of the two epithelial phenotypic proteins (EpCAM and MUC1) on tumor-derived sEVs simultaneously, can our designed lanthanide luminescence probe precursors then assemble to form the AND gate for photoluminescence detection. Consequently, the generated AND gate photoluminescence provided time-resolved luminescence for a wide cancerous sEV linear detection range of 6.0 × 104-6.0 × 109 particles per mL, with a calculated detection limitation of 1.42 × 102 particles per mL. Furthermore, the AND gate photoluminescence can significantly distinguish epithelial cancer patients from healthy controls, displaying its great potential for accurate and noninvasive cancer diagnosis.