Development of catalysts with high activities for biomedicine and environmental protection has attracted widespread attention. In this study, B-doped graphite carbon nitride (B-g-C3N4) was obtained by polycondensation of melamine with boron oxide as a dopant. The single-atom Fe nanozymes supported on B-g-C3N4 (SA Fe/B-g-C3N4) were prepared via ion exchange, followed by annealing at elevated temperatures. Synchrotron X-ray absorption spectroscopy and scanning transmission electron microscopy investigations demonstrated the successful synthesis of single-atom Fe catalysts. The obtained SA Fe/B-g-C3N4 showed good peroxidase-like activity in the presence of H2O2. Density functional theory calculations verified the promotion effect of B doping on catalytic reactions. Based on these findings, SA Fe/B-g-C3N4 was constructed as a 3-minute colorimetric sensor in response to sulfide anion concentration with a detection limit of 0.57 µmol L−1. The feasibility of such protocol in S2− detection was verified in tap water and sea water.