In this work we have fabricated a glutathione-S-transferase based amperometric biosensor for pesticides by immobilizing the enzyme on platinum electrode using graphene oxide-gelatin matrix, evaluated various biosensor parameters, applied the same for detection and quantification of four different classes of pesticides and validated the biosensor results with GC-MS analysis. The enzyme immobilization was confirmed through scanning electron microscopy, electrochemical impedance spectroscopy, cyclic voltammetry and chronoamperometry. The apparent Michaelis-Menten constant for the immobilized glutathione-S-transferase in the said matrix was found to be 0.083 mmol L-1 and 0.15 mmol L-1 respectively for glutathione and 1-Chloro-2,4-dinitrobenzene. Substrate specificity found to be 2.56 x 10(7) s(-1) M-1 for glutathione and 2.15 x 10 7 s(-1) M-1 for 1-Chloro-2,4-dinitrobenzene. Pesticide analysis was done in 25% methanol solution. The biosensor is a promising new tool for pesticide analysis as it can be applied for analysis of a broad spectrum of pesticides covering at least six different classes namely - benzamidazole, organochlorine, organothiophosphate, organocarbamate, polyphenol and pyrethroid.