1,5-anhydroglucitol (1,5-AG) is an important indicator in the early detection of diabetes. In this study, a high-performance light addressable potentiometric sensor (LAPS) for the detection of 1,5-AG based on reduced graphene oxide-polyacrylamide-ferrocene/gold nanoparticles (rGO-PAM-Fc/AuNPs) as sensing membrane and pyranose oxidase (PROD) as the specific recognition substance was constructed. Among them, rGO-PAM-Fc/AuNPs modification LAPS chip can not only effectively increase the specific surface area and adsorb more PROD, but also enhance the capacitive effect of LAPS and improve the detection sensitivity. 1,5-AG is hydrolyzed by PROD and produces hydrogen peroxide (H2O2) that can oxidize divalent ferrocene ions in the rGO-PAM-Fc composite to trivalent ferrocene ions, therefore leads to a change in the membrane potential on the surface of the LAPS chip and causes a shift in the I-V curve, which reflect the change of 1,5-AG concentration. The designed LAPS had a good linearity with 1,5-AG concentration in the range of 100.0–1000.0 μg/mL with a correlation coefficient of 0.99068. The sensitivity was calculated to be 0.1908 mV/μg/mL and a limit of detection (LOD) of 21.74 μg/mL was obtained using three signal-to-noise ratio (S/N = 3). Also, the 1,5-AG biosensor has good specificity, reproducibility and stability, and can be used in human serum samples with the recoveries of 93.62 %–114.52 %. This work provides a highly sensitive, portable and low-cost assay for the detection of 1,5-AG. Prime novelty statementHerein, a high-performance light addressable potentiometric sensor (LAPS) for the detection of 1,5-AG based on reduced graphene oxide-polyacrylamide-ferrocene/gold nanoparticles (rGO-PAM-Fc/AuNPs) as sensing membrane and pyranose oxidase (PROD) as the specific recognition substance was constructed. Among them, rGO-PAM-Fc/AuNPs modification LAPS chip can not only effectively increase the specific surface area and adsorb more PROD, but also enhance the capacitive effect of LAPS and improve the detection sensitivity. 1,5-AG is hydrolyzed by PROD and produces hydrogen peroxide (H2O2) that can oxidize divalent ferrocene ions in the rGO-PAM-Fc composite to trivalent ferrocene ions, therefore leads to a change in the membrane potential on the surface of the LAPS chip and causes a shift in the I-V curve, which reflect the change of 1,5-AG concentration. The designed LAPS had a good linearity with 1,5-AG concentration in the range of 100.0–1000.0 μg/mL with a correlation coefficient of 0.99068. The sensitivity was calculated to be 0.1908 mV/μg/mL and a limit of detection (LOD) of 21.74 μg/mL was obtained using three signal-to-noise ratio (S/N = 3). Also, the 1,5-AG biosensor has good specificity, reproducibility and stability, and can be used in human serum samples with the recoveries of 93.62 %–114.52 %. This work provides a highly sensitive, portable and low-cost assay for the detection of 1,5-AG.