An amperometric sensor with attractive sensing behavior was prepared by using solid carbon paste as substrate for electropolymerization of 2-amino-5-mercapto-1,3,4-thiadiazole (AMT). Morphology, polymerization mechanism and charge-transfer performance of the poly-AMT (PAMT) film on the composite substrate surface were investigated by using SEM, in situ UV–vis spectroelectrochemistry and electrochemical impedance spectrum methods. The results indicate that the PAMT film covered the wax portion prior to the graphite grains on the substrate surface, and the resulting sensor showed remarkable decrease in charge-transfer resistance with the increase in the amount of deposited PAMT. At the physiological pH the sensor gave sensitive and well separated voltammetric signals for two bioflavonoid isomers with the same electro-active moiety, luteolin and fisetin. The sensor achieved amperometric sensitivities of 1.41nAnM −1 cm −2 to luteolin and 0.648nAnM −1 cm −2 to fisetin in a linear range of 10–700nM, with the lowest detection limits of 3nM luteolin and 5nM fisetin, respectively. The study demonstrates that the solid carbon paste is a superior substrate material for PAMT film sensor.