Prostate specific membrane antigen (PSMA)-positive exosomes have the potential to serve as highly sensitive biomarkers for prostate cancer detection. Herein, a sensitive electrochemical biosensor for the ultrasensitive detection of PSMA-positive exosomes has been constructed based on a peptide-templated AgNPs nanoprobe. In this work, PSMA-specific binding peptides immobilized on a gold electrode were responsible for prostate cancer-derived exosomes capturing. Well-designed peptide (CCY- LWYIKC) serves a dual role: as a signal probe and as a recognizer in the exosomes-identification process. Specifically, LWYIKC bind to cholesterol at the exosome membranes, and CCY function as peptide templates to host a large number of silver nanoparticles, leading to a strong electrochemical signal. Thus, the concentration of exosomes can be quantified via electrochemical signal. This innovative method displayed a wide detection range of 102 to 108 particles/μL and a detection limit as low as 37 particles/μL. Notably, the method has shown outstanding performance when validated using clinical samples, suggesting its potential for clinical applications.