PurposeIn this paper, hybrid multienzyme biosensor system, which detects analyte through molecular conversion into signal response, is analyzed and presented. The biokinetic effects of pertinent parameters such as Michaelis–Menten constant, inhibitor inhibition and substrate inhibition modulus on biochemical reactions are investigated.Design/methodology/approachBiochemical reaction models are described by five nonlinear equations for bisubstrate amperometric system analyzed adopting the regular perturbation method.FindingsResults obtained reveal that increasing Michaelis–Menten constant of oxygen causes a significant decrease in hydrogen peroxide concentration while increasing Michaelis–Menten constant of glucose shows increasing effect on oxygen concentration. Hence, results obtained from this work serve as reference for further analysis of concentration models and offer useful insight to relevant applications such as food safety, environmental and biomedical applications.Practical implicationsThis work serves as reference for further analysis of concentration models and offers useful insight to relevant applications such as food safety, environmental and biomedical applications.Originality/valueThis paper examines the effect of biokinetic parameters on the concentration of the hybrid multienzyme biosensor. Here the effects of parameters such as inhibitor inhibition, substrate inhibition and Michealis–Menten were investigated on substrate, inhibition and product concentrations. It is illustrated from result that inhibitor parameter slows enzymatic catalytic reaction while substrate enhances reaction. This study applied approximate analytical scheme to investigate the biokinetic effects, adopting the regular perturbation scheme.