We report here on novel biosensors for the highly sensitive and specific enzymatic analysis of glycated hemoglobin (HbA1c), which we measured primarily to identify three‐month average plasma glucose concentrations. Hydrogen peroxide (H2O2) was generated in this enzymatic measuring of glycated hemoglobin from oxidative cleavage of fructosyl valine by fructosyl amino acid oxidase, and we then used the generated hydrogen peroxide as a reducing agent of gold (III) ion to synthesize gold (0) as a nanoparticle. We were able to measure the gold nanoparticles obtained from this novel approach using colorimetry under a UV–VIS spectrophotometer. For this colorimetric detection, we analyzed 96‐well plates for glycated hemoglobin, and this system worked very well, with a detection limit of 0.142% and coefficient of variation below 10%. Finally, this method can measure glycated hemoglobin in patients with diabetes.