A fluorometric method for assaying the activity of horseradish peroxidase (HRP) in organic media has been developed. This method is designed on the basis of the disparity in the spectral properties of substrates and corresponding resultant polymers. It monitors the fluorescence quenching of substrate during enzymatic catalysis, and works efficiently in a number of organic media (such as dioxane-water mixture, acetone-water mixture, and alcohol-water mixture, and so forth) toward many substrates. This assay is simpler, more rapid, and more convenient compared with the commonly used HPLC method. It is qualitatively reproducible and can also be used for quantitative calculation of the substrate conversion.