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This work describes a novel affinity peptide–antibody sandwich electrochemical strategy for the ultrasensitive detection of prostate-specific antigen (PSA). Herein, polydopamine-coated boron-doped carbon nitride (Au@PDA@BCN) was synthesized and used as a sensing platform to anchor gold nanoparticles and immobilize primary antibody. Meanwhile, AuPt metallic nanoparticle and manganese dioxide (MnO2)-functionalized covalent organic frameworks (AuPt@MnO2@COF) was facilely synthesized to serve as a nanocatalyst and ordered nanopore for the enrichment and amplification of signal molecules (methylene blue, MB). PSA affinity peptide was bound to AuPt@MnO2@COF to form Pep/MB/AuPt@MnO2@COF nanocomposites (probe). The peptide–PSA–antibody sandwich biosensor was constructed, and the redox signal of MB was measured with the existence of PSA. The fabricated sensor exhibited a linear response (0.00005–10 ng mL−1) with a low detection limit of 16.7 fg mL−1 under the optimum condition. Additionally, the sensor showed an excellent selectivity, ideal repeatability, and good stability for PSA detection in real samples. Furthermore, the porous structure of COF can enrich more MB molecules and increase the sensitivity of the biosensor. This study provides an efficient and ultrasensitive strategy for PSA detection and broadens the use of organic/inorganic porous nanocomposite in biosensing.