Isoniazid (INZ) is an effective anti -tuberculosis drug that has been widely used for chemotherapy of tuberculosis. Development of new and simple electroanalytical method for routine analysis of INZ is a continued research interest in the field of analytical and pharmaceutical chemistry. Although there have been several electroanalytical reports, flow injection analysis coupled electrochemical detection of INZ, which is an advanced electrochemical technique useful for practical applications, is rarely reported in the literature. Instability problem associated with the underlying electrode by the amino -functional group of the INZ is the prime reason for the limitation. Herein, we report a gold nanoparticles decorated carbon nanofibers-chitosan modified carbon screen printed electrode, designated as Au-nano@CNF-CH1T/SPE, prepared by drop-casting of Au3+ ion directly on CNF-CHIT/SPE underlying electrode followed by electrochemical potential cycling, as an elegant electrochemical detector system for high stable FIA of INZ in pH 7 phosphate buffer solution. The modified electrode showed about fifty times higher electrocatalytic current and 700 mV reduction in the over -potential values than that of a polycrystalline gold modified electrode towards INZ. Under an optimal hydrodynamic FIA condition, the present ECD showed a wide linear range from 1 mu M to 1 mM with regression coefficient and sensitivity values of 0.9958 and 16.1 nA mu M-1 respectively. The calculated limit of detection and limit of quantification values are 172 nM (i.e., 472 pg/20 mu L) and 570 nM (i.e., 1.57 ug/20 mu l,), respectively. The present FIA-ECD was successfully applied for the real sample analysis by detecting INZ in two pharmaceutical formulations with satisfactory good recovery values.