Abstract: Scopoletin, 7-hydroxy-5-methoxycoumarin, has been found to be a moderate inhibitor for aldehyde oxidase, a molybdo-flavoenzymes. It inhibits the release of superoxide anion more efficiently than hydrogen peroxide or substrate oxidation. Statistically, the inhibition of three-specific aldehyde oxidase substrates was significant (p<0.005) in the presence of 100 μM Scopoletin. A progressive and non-competitive inhibition has been observed with inhibitor constant (Ki) value of 57.2 ±3.3 μM using indole-3-aldehyde and oxygen as a substrate and electron acceptor, respectively. A specific site for interaction between Scopoletin and aldehyde oxidase has been proposed and its effect on the reactive oxygen species production has been discussed. The effect of Scopoletin on aldehyde oxidase activity has been compared with that of traditional and specific inhibitors, such as menadione.