A real-time kinase assay method based on a ratiometric fluorescence probe that can be applied to various small-molecule kinases is described herein. The probe can trace the reversible interchange of ATP and ADP, which is a common phenomenon in most small-molecule kinase reactions, by a ratiometric fluorescence change. This property facilitates the monitoring of phosphorylation and dephosphorylation in small-molecule kinases, whereas most of the existing methods focus on one of these reactions. To prove the applicability of this method for small-molecule kinase assays, hexokinase and creatine kinase, which phosphorylate and dephosphorylate substrates, respectively, were analyzed. The ratiometric fluorescence change was correlated with the enzyme activity, and the inhibition efficiencies of the well-known inhibitors, N-benzoyl-D-glucosamine and iodoacetamide, were also monitored. Notably, the change in fluorescence can be observed with a simple light source by the naked eye.