Bacterial infection is one of the major causes of human death worldwide. To prevent bacterial infectious diseases from spreading, it is of critical importance to develop convenient, ultrasensitive, and cost-efficient methods for bacteria detection. Here, an electrochemical detector of a functional two-dimensional (2D) metal-organic framework (MOF) nanozyme was developed for the sensitive detection of pathogenic Staphylococcus aureus. A dual recognition strategy consisting of vancomycin and anti-S. aureus antibody was proposed to specifically anchor S. aureus. The 2D MOFs with excellent peroxidase-like activity can efficiently catalyze o-phenylenediamine to 2,2-diaminoazobenzene, which is an ideal electrochemical signal readout for monitoring the bacteria concentration. Under optimal conditions, the present bioassay provides a wide detection range of 10-7.5 × 107 colony-forming units CFU/mL with a detection limit of 6 CFU/mL, which is better than most of the previous reports. In addition, the established electrochemical sensor can selectively and accurately identify S. aureus in the presence of other bacteria. The present work provides a new pathway for sensitive and selective detection of S. aureus and presents a promising potential in the realm of clinical diagnosis.