Small extracellular vesicles (sEVs), often referred to as exosomes, are potential biomarkers for noninvasive cancer diagnosis. However, because of their phenotype heterogeneity, precise detection of tumor-derived sEVs is a great challenge. Herein, a dual-aptamer-assisted AND logic gate was fabricated for sensitive electrochemical detection of tumor-derived sEVs based on a cyclic enzymatic signal amplification strategy. Four different tumor-derived sEVs were used to verify the feasibility of the AND logic gate, and CCRF-CEM sEVs were successfully detected by this assay. The electrochemical assay shows a good linear response from 4 × 103 to 8 × 107 particles/μL, with a detection limit of 920 particles/μL, for CCRF-CEM sEVs, indicating potential application in accurate cancer diagnostics.