Chlorine dioxide is a disinfectant used worldwide. In this article, a new enzymatic method for the determination of chlorine dioxide has been developed. This rapid spectophotometric assay is able to detect from 0.2 to 4 mg/liter of chlorine dioxide. The method is based on the capacity of horseradish peroxidase to decolorize reactive yellow 17 in the presence of chlorine dioxide. The effects of several compounds on the assay have been determined. Except sodium hypochlorite, no interference was detected with 18 compounds including chlorides, sulfates, carbohydrates, amino acids, proteins, and organics. The biochemical method is faster and easier than the previous volumetric, amperometric, and colorimetric methods which are laborious and time-consuming.