The invention provides a determination method of ammonia content in cigarette mainstream smoke. According to the determination method provided by the invention, by taking Li+ as an internal standard substance, ion chromatography detection of the ammonia content in the cigarette mainstream smoke is realized. The determination method provided by the invention avoids the interference of the environmental background to an electrical conductivity detector, under different environmental backgrounds, for different cigarette samples, the ammonia content in the cigarette mainstream smoke can be quantitatively obtained by adopting the same standard curve, and detection results have high accuracy and repeatability. Experimental results show that at different environment temperatures, the detection results obtained by the determination method provided by the invention have high repeatability, so that the stability and accuracy of detection are improved.