Bovine serum albumin (BSA) was firstly implemented as an effective sensitivity enhancer for a peptide-based amperometric biosensor for the ultrasensitive detection of prostate specific antigen (PSA). A porous and conductive substrate of chitosan-lead ferrocyanide-(poly(diallyldimethylammonium chloride)-graphene oxide) was in-situ generated on a glassy carbon electrode (GCE), in which Pb2Fe(CN)6 served as a novel redox species with strong current signal at −0.46 V (vs. Ag/AgCl). Poly(diallyldimethylammonium chloride)-graphene oxide was applied to improve conductivity of the substrate. After adsorbing Pb2+ for signal amplification, chitosan provided active sites to simultaneously immobilize peptides and 1-aminopropyl-3-methylimidazolium chloride by glutaraldehyde. To enhance the sensitivity, BSA was chemically linked to the immobilized peptide, behaving as a serious decrease of current signal for BSA hindering the electron transfer. The dramatic increase of current signal of the biosensor was obtained by PSA cleaving the immobilized BSA-peptide. The proposed biosensor exhibited a detection limit of 1fgmL−1 for PSA and its sensitivity was seven-fold higher than previous works. •BSA was firstly implemented as an effective sensitivity enhancer for the peptide-based amperometric biosensor.•Multiple amplification strategies were developed for the amperometric biosensor for PSA.•Chitosan-Pb2Fe(CN)6-PDDA-GO as a new redox species was used as substrate.