A coloring reaction of leucomarachite green with hemoglobin (Hb) and horseradish peroxidase (HRP) under various concentrations of hydrogen peroxide was examined. The optimum concentration of hydrogen peroxide at the reaction with HRP was 0.25 mM, being lower than that with Hb. With 75 mM of hydrogen peroxide, the coloring reaction of leucomarachite green with HRP was not observed and the spectrum of HRP with 75 mM of hydrogen peroxide showed “Compound III”, which is known as an inactive-intermediate of HRP produced by excess hydrogen peroxide. These results suggest that HRP was inactivated under the concentration of hydrogen peroxide (180 mM) used at a preliminary test for bloodstain. Thus, the concentration of hydrogen peroxide used in the preliminary test may be related to the specificity of leucomarachite green test for Hb.