We describe a highly sensitive and selective peptide-based biosensor for the prostate-specific antigen (PSA). The biotinylated peptide biotin-EHSSKLQKC served as a molecular recognition element. It was self-assembled on the surface of the wells of a 96-well microtiter plate modified with gold nanoparticles (AuNPs). In parallel, streptavidin-coated magnetic beads (strep-MBs) were modified with the enzyme invertase and then added to the peptide-modified wells upon which the modified strep-MBs bind to the peptide via biotin/streptavidin interaction. If a sample containing PSA is placed in the well, PSA will cause the cleavage of the peptide, and a respective quantity of invertase-modified strep-MBs will be released. The supernatant containing the invertase-modified strep-MBs is taken out and sucrose is added which is enzymatically cleaved by invertase. The concentration of the glucose formed after 1 h is quantified with a personal glucose meter. It is linearly related to the concentration of PSA in the range from 80 pg to 7 ng∙mL−1. The detection limit is 30 pg∙mL−1 and the relative standard deviation is 3.7 % (at a level of 500 pg∙mL−1 and for n = 7). The method was successfully applied to the determination of PSA in spiked real human urine. Due to its simplicity, sensitivity and selectivity, this bioassay offers a promising approach to the detection of PSA and other biomolecules.