Binding of nitric oxide to horseradish peroxidase (HRP) has been investigated by absorption spectrometry in 0.2M anaerobic phosphate buffer solution (pH 7.4). Based on this binding equilibrium, a model equation for evaluating the binding constant of nitric oxide to HRP is developed and the binding constant is calculated to be (1.55±0.06)×10 4 M −1 , indicating that HRP can form a stable complex with nitric oxide. The type of inhibition by nitric oxide is validated on the basis of studying initial reaction rates of HRP-catalyzed oxidation of guaiacol in the presence of hydrogen peroxide and nitric oxide. The inhibition mechanism is found to follow an apparent non-competitive inhibition by Lineweaver–Burk method. Based on this kinetic mechanism, the binding constant is also calculated to be (5.22±0.06)×10 4 M −1 . The values of the binding constant determined by the two methods are almost identical. The non-competitive inhibition model is also applicable to studying the effect of nitric oxide on other metalloenzymes, which catalyze the two-substrate reaction with the “ping-pong” mechanism.