A novel peptide-based electrochemiluminescent（ECL）method for the determination of prostate-specific antigen（PSA）was developed based on the target-induced cleavage of homogeneous specific polypeptide with gold nanoparticles（AuNPs） to overcome drawbacks from probes directly immobilized on electrodes.A specific peptide CHSSKLQK was employed as a molecular recognition element，and ruthenium（Ⅱ）complexes（Ru1）were used as ECL emitting species.A site-specific enzymatic-cleavage-reaction-based biosensor using Ru1/peptide-conjugated gold nanoparticle complexes（AuNPs-peptide-Ru1）was developed.When PSA was added into the solution containing AuNPs-peptide-Ru1，PSA specifically recognized and cleaved the sequence of the peptides attached to the AuNPs.As a result，Ru1 were separated from the AuNPs and resulted in the increase of ECL intensity in the presence of co-reactant tripropylamine.Results showed that the increased ECL intensity was directly linear to the logarithm of PSA concentration in range of 5.0×10-12-1.0×10-9 g/mL with a detection limit of 2.0×10-12 g/mL.The method was applied in the detection of clinical serum samples，and the results were consistent with those obtained by CL method.With the advantages of high sensitivity and good reproducibility，this method provides a promising strategy for the sensitive detection on protease.