Nanoparticle-based assays offer rapid, sensitive, and real-time detection of biomolecules and are increasingly applied in bioanalytical sensing. However, due to the widespread applications of alkaline phosphatases in medicine, research and industry have a preference for this rapid, nanobased sensitive assay for detecting alkaline phosphatase activity. In this study, a simple and cost-effective nanobioassay was developed for the detection of alkaline phosphatase activity using green-synthesized silver nanoparticles. The assay is based on the ability of the enzyme substrate adenosine triphosphate (ATP) to inhibit salt-induced aggregation of silver nanoparticles. Synthesised silver nanoparticles were used for the bioassay, which served as both reducing and capping agents, avoiding the use of hazardous chemicals. Nanoparticle formation was confirmed by a characteristic surface plasmon resonance peak at ~420 nm in UV–Vis spectra, and dynamic light scattering analysis revealed an average particle size of ~80 nm.