Numerous assays based on peroxidase activity have been developed for the detection of analytes due to the various optical peroxidase substrates. However, most substrates are sensitive to light and pH and are over-oxidized in the presence of excess H₂O₂. In this study, 2-((6-methoxynaphthalen-2-yl)methylene)-1,1-dimethylhydrazine (MNDH), a fluorescent peroxidase substrate prepared from naphthalene-based aldehyde N,N-dimethylhydrazone, was developed. MNDH showed quantitative fluorescence changes with respect to the H₂O₂ concentration in the presence of horseradish peroxidase (HRP), and the MNDH/HRP assay showed no changes in fluorescence caused by over-oxidation in the presence of excess H₂O₂. Further, MNDH was thermo- and photostable. Additionally, the assay could be operated over a considerably wide pH range, from acidic to neutral. Moreover, MNDH can be used to detect glucose quantitatively in human serum samples by using an enzyme cascade assay system.