Monitoring and control of penicillin (PCN) in the environment are critically important for reducing the emergence and dissemination of antibiotic-resistant bacteria, ensuring food safety, and safeguarding human health. In this study, we introduce a novel and facile strategy for modulating the catalytic activity of bimetallic nanozymes through surface ligand engineering. Experimental activity assays and theoretical model calculations confirm that Ni-Pt nanozymes coated with polyallylamine hydrochloride (PAM) exhibit significantly enhanced peroxidase (POD)-like activity compared to their counterparts coated with conventional polyvinyl pyrrolidone (PVP) and polystyrene sulfonate (PSS). Leveraging the potent reactivity between PCN and the hydroxyl radicals (∙OH) generated by Ni-Pt@PAM catalytic system, we have developed a colorimetric sensor that directly detects PCN, demonstrating a linear response range from 0.5 to 10 μM and detection limit as low as 13.5 nM. Additionally, a fluorescent sensing platform utilizing self-synthesized fluorophore Rho-Si based on inner filter effect (IFE), has been successfully established. These two sensing platforms, capable of generating both colorimetric and fluorescent signals, enable sensitive, rapid, and straightforward detection of PCN in water samples. This work not only offers an innovative approach for tuning the POD-like activity of Ni-Pt bimetallic nanozymes but also presents a promising avenue for their application in PCN monitoring.