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In applicative biosensor technology, mathematical modeling plays an indispensable role in explaining the transport of electrical signals by analyzing the binding behavior of the biochemical enzyme inhibitors to the target molecule. The biosensors are extensively used in clinical diagnostics, drug detention, food analysis and environment monitoring because they are highly sensitive, reliable and relatively cheap. Dynamic mathematical models used for biological investigation serve the purpose efficiently with very reasonable outcomes. In this study, a time-independent mathematical model for biosensor enzyme–substrate–inhibitor system under uncompetitive inhibition based on the nonlinear diffusion equations taking into consideration the kinetic rate constants and the initial concentrations of enzyme, substrate and inhibitor has been formulated and solved analytically using variational iteration method (VIM). The reliability and accuracy has been proved by comparing our results with the solution obtained by standard VIM. Chosen biosensors showed desirable sensitivity, selectivity and potential for application on real samples. They are frequently made to prevent interference from undesirable components that are present in the monitored system. The VIM is effectively and easily used to obtain solution of nonlinear equations accurately. Further, the solution has been discussed exhaustively for different values of reaction parameters avoiding linearization and unrealistic assumptions and the results obtained significantly agree with existing literature.