Abstract：It was found that strong chemiluminescence (CL) was directly produced by the reaction of isoniazid with luminol in the presence of gold nanoparticles (AuNPs) in an alkaline solution. Based on this finding, a simple and sensitive CL method has been developed for the determination of isoniazid. The reagent concentrations, including luminol concentration, NaOH concentration and AuNPs concentration, were optimized with the aid of Box-Behnken design and response surface methodology. The method allows the measurement of 5.0×10－9～3.0×10－7 g/mL isoniazid with a detection limit of 4.1×10－9 g/mL. The relative standard deviation is 1.3% for 1.0×10－7 g/mL isonazid solution in 11 repeated measurements. The proposed method was applied to the analysis of isoniazid in tablets, injections and in spiked human blood serums. The possible CL reaction mechanism was also discussed by the study of the UV-vis spectrum, the CL spectrum, and the effect of dissolved oxygen and reactive oxygen species scavengers on the reaction.