Abstract: A method based on single particle inductively coupled plasma mass spectrometry (spICP-MS) was established to accurately quantify the particle number concentration of polydisperse gold nanoparticles (AuNPs) sample with two sizes of 30 nm and 60 nm. The effect of carrier gas flow rate and sampling depth on the size resolution, as well as the effect of acquisition time and dwell time on the measured particle number concentration were investigated. The results showed that the optimized carrier gas flow rate could significantly improve the size resolution, while the impact of the sampling depth was relatively negligible. The highest size resolution was obtained when the carrier gas flow rate was 0.8 L/min and the sampling depth was 9 mm, and the extended acquisition time could effectively reduce the relative standard deviation (RSD) of the result. When the acquisition time was increased to 180 s, the particle number concentration of 30 nm and 60 nm AuNPs in the polydisperse sample was lower than 5% (n=3). When the dwell time was selected as 0.1 ms, the result was consistent with the prepared value, and the particulate and ionic Au could be entirely separated. Under the optimized conditions, the detection limits of particle size and particle number concentration were 10 nm and 49 NPs/g, respectively. The established method was employed successfully to quantify the polydisperse sample with different mixing ratios, and the results were consistent with the standard values, proving the reliability of the method. Finally, this method was successfully applied to tap water, spring water and lake water, and the recovery rates of the three water samples were satisfied in the range of 80%-120%. The method has the advantages of high size resolution, better precision and low ion interference, which is an accurate method for the quantification of polydisperse nanoparticles in the environmental matrix.