Abstract: An efficient and stable method was developed for quantitative analysis of ergothioneine (EGT) by high performance liquid chromatography (HPLC). The method was carried out on Zorbax SB-Aq Column with elution A (the volume ratio of urtrapure water vs methanol was 99:1, adjusted to pH5 by 20% of acetic acid and 50% of ammomnia and elution B (methanol) as mobile phase at 0.7 mL/min. An UV-VIS detector with a wavelength of 257 nm was employed. The injection volumn was 5 μL, with the column temperature being 30℃. The gradient elution program makes the method more stable and prolong utility time of the chromatographic column. Based on the results of HPLC and HPLC-MS, we detected the specificity of this method was good. The limit of detection (LOD) and limit of quantification (LOQ) were 0.045 and 0.900 μg/L. Good linearity (correlation coefficient R²=0.9991) could be achieved for EGT quantification at the range of 2.5 to 1000 mg/L.The RSD of precision, stability in 12 h and stability in 14 d were 0.33%, 1.89%, and 0.026% respectively. Meanwhile, the average recoveries of EGT were within 92.17%~93.92%. The method was simple, rapid, stable and accurate, which could be used in the research and application of EGT biosynthesis