A new method to assay horseradish peroxidase(HRP)catalytic activity of coupling phenols and aromatic amines in nonaqueous media with fluorescence spectrophotometer was developed based on that the fluorescence emission spectra of the substrates and corresponding product polymers were not overlapped.This method could detect the activity of HRP for different substrates in various nonqueous reaction systems(such as dioxane-water mixture,acetone-water mixture) by measuring the quenching of the substrate fluorescence.The present work offered a fast and convenient method for determining the activity of HRP in nonqueous media.This assay is qualitatively reproducible.It can also be used for quantitative calculation of the substrate conversion.