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• Electrochemical impedimetric biosensor for umami substance detection was developed. • Electrochemical impedance spectroscopy enhanced T1R1 biosensor performance. • T1R1 biosensor had low detection limits for monosodium glutamate and inosine-5′-monophosphate. • T1R1 biosensor had wide linear ranges for monosodium glutamate and inosine-5′-monophosphate. Umami substances can provide a palatable flavour for food. In this study, an electrochemical impedimetric biosensor was developed for detecting umami substances. This biosensor was fabricated by immobilising T1R1 onto AuNPs/reduced graphene oxide/chitosan which was in advance electro-deposited onto a glassy carbon electrode. The evaluation by the electrochemical impedance spectrum method showed that the T1R1 biosensor performed well with low detection limits and wide linear ranges. Under the optimised incubation time (60 s), the electrochemical response was linearly related to the concentrations of the detected targets monosodium glutamate and inosine-5′-monophosphate within their respective linear range of 10-14 to 10-9 M and 10-16 to 10-13 M. The low detection limit of monosodium glutamate and inosine-5′-monophosphate was 10−15 M and 10−16 M, respectively. Moreover, the T1R1 biosensor exhibited high specificity to umami substances even in the real food sample. The developed biosensor still retained 89.24% signal intensity after 6-day storage, exhibiting a desirable storability. [ABSTRACT FROM AUTHOR]