Monitoring cefoperazone (CFZ) in milk, serum, and pharmaceutical injections is essential for ensuring consumer safety, proper therapeutic exposure, and product quality. Residue control in milk protects against antimicrobial risks, while serum monitoring prevents toxicity or underdosing. Quality assessment of injections ensures formulation integrity throughout production. In this study, molybdenum–boron/sulfur co-doped carbon dots (Mo/B, N, S@CDs) were synthesized as a fluorescent nanozyme with strong peroxidase-like activity and a photoluminescence quantum yield of 43.55 %. In the presence of H₂O₂, the nanozyme catalyzed the oxidative coupling of 4-aminoantipyrine (4-AAP) with CFZ, producing a red quinoneimine dye with an absorption peak at 515 nm. This enabled colorimetric detection, with absorbance increasing proportionally to CFZ concentration. Concurrently, the dye quenched the green fluorescence of Mo/B, N, S@CDs at 530 nm, providing a sensitive fluorometric mode. The exceptional performance stems from synergistic Mo/B, N, S co-doping, which enhances peroxidase-like activity, optimizes electron transfer, and enables dual-mode signal amplification. Under optimized conditions, the colorimetric and fluorometric methods offered linear ranges of 0.1–500 μM and 0.01–500 μM with detection limits of 0.018 μM and 0.008 μM, respectively. The dual-mode platform performed reliably in milk, serum, and injection samples, yielding recoveries of 98.0–105.7 % and RSDs below 4.00 %, demonstrating its suitability for routine CFZ monitoring.