A novel electrogenerated chemiluminescence (ECL) bioanalytic system based on biocleavage of a ECL probe and homogeneous detection was designed and utilized for the first time for highly sensitive quantification of proteases to overcome drawbacks from probes directly immobilized on electrodes and commercial ECL biosystems, based on bioaffinity reactions. Prostate-specific antigen (PSA) was taken as a model analyte and ruthenium complex-tagged specific peptide (CHSSKLQK) was designed as an ECL probe (peptide-Ru1). ECL bioconjugated magnetic beads were synthesized through a simple solid-phase synthesis. When analyte PSA was introduced into the suspension of ECL bioconjugated magnetic beads, a biocleavage of the peptide occurred and the cleaved Ru1 part was released from the magnetic beads. ECL measurement was carried out in the presence of co-reactant tripropylamine, using two models. One is homogeneous ECL detection on a bare graphite pencil electrode (PGE), and the other is enriching ECL detection after the cleaved Ru1 part of the peptide was concentrated into the surface film of Nafion/gold nanoparticles modified PGE (AuNPs/Nafion/PGE). The extremely low detection limit of 80 fg/mL and high reproducibility (relative standard deviation (RSD) of 5.4% for six measurements of 0.5 pg/mL) for the detection of PSA were achieved at AuNPs/Nafion/PGE. This work demonstrates that the bioanalytic system designed can not only quantify proteases with high sensitivity and selectivity, but also diminish the complicated electrode process and improve the reproducibility by conducting the biocleavage and transduction steps at different surfaces. It can be easily extended for ECL analysis of other proteases in this system and other detection techniques, including optics and electrochemistry.