Polyanionic phosphate derivatives, ATP and phosphopeptide, were shown to induce the supramolecular assembly of cationic alkynylplatinum(II) terpyridine complexes via the formation of metal–metal and π–π stacking interactions with significant UV-vis, near-infrared (NIR) emission and circular dichroism spectral changes. The induced supramolecular assembly behaviours of these platinum(II) complexes were shown to be sensitive towards microenvironmental changes as well as the negative charge densities of the phosphate derivatives, providing the complexes with the capability of distinguishing the target substrates from their respective metabolic products by sole structural differences. Through the monitoring of the NIR emission spectral changes that are dependent on the extent of the assembly/disassembly of the platinum(II) complexes, the conversion of the target substrates catalyzed by ATPase, v-Src kinase and alkaline phosphatase could be signaled and probed on a real-time basis. The kinetic parameters of the enzymatic activities have also been determined.