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Prostate cancer-derived exosomes have important potential as biomarkers for diagnosis and treatment of prostate cancer. But such specific exosomes towards clinical application remains problematic due to their comparatively low concentration in relation to other constituents of blood. Additionally, the presence of particles in blood that share a similar size with exosomes adds to the complexity of their selective and sensitive detection. Consequently, the detection of exosomes derived from prostate cancer in intricate biological settings necessitates the implementation of highly sensitive and specific biosensors. Herein, we report an electrochemical biosensor for prostate cancer-derived exosomes detection, with two-level selectivity achieved through a sandwich structure involving specific peptides and two-level amplification utilizing the combination of biotin-streptavidin linkage and G-quadruplex hemin mimetic peroxidase to enhance the sensitivity. Evaluation of PSMA positive exosomes at various concentrations demonstrates a remarkable limit of detection as low as 26 particles/μL, as well as an excellent linear sensor response spanning from 1.0 × 102 to 1.0 × 107 particles/μL. Compared to the enzymatic biosensor, this biosensor proves more versatile without a label or enzyme, and may be more promising for clinical applications.