Aims To evaluate the analytical performance of a fully automatic iFlash anti-Müllerian hormone immunoassay (YHLO Biotech) and establish age-specific reference range for anti-Müllerian hormone in Chinese women based on a multicentre population study. Design and method iFlash anti-Müllerian hormone assay was evaluated for sensitivity, imprecision, serial dilution for linearity, impact of sample type, storage and stability. Method comparison of iFlash anti-Müllerian hormone with Elecsys anti-Müllerian hormone was studied. Reference intervals in healthy females were established for iFlash anti-Müllerian hormone. Results The limit of blank and limit of detection were below 0.001 ng/mL and 0.02 ng/mL, respectively. Assay sensitivity defined as limit of quantitation was 0.08 ng/mL. The assay imprecision was similar at low and high concentration being 3.1% and 3.2%, respectively. The linearity was observed to be between 0.02 ng/mL and 27.22 ng/mL. The stability of AMH was most at −80°C and up to seven days at 4°C, −20°C. No significant difference was observed for anti-Müllerian hormone among different sample types. An excellent agreement of anti-Müllerian hormone concentration was found in 180 samples analysed by iFlash and Roche; the correlation coefficient was 0.975 and regression slope of 1.009. The AMH reference intervals for Chinese women aged between the ages of 20 and 49 years with five-year intervals were 1.20–10.21 ng/mL, 1.14–9.17 ng/mL, 0.55–8.18 ng/mL, 0.25–7.02 ng/mL, 0.07–4.59 ng/mL and 0.01–2.11 ng/mL, respectively (1 ng/mL = 7.14 pmol/L). Conclusion The fully automated iFlash anti-Müllerian hormone immunoassay demonstrates excellent analytical performance. Consequently, the availability of iFlash anti-Müllerian hormone assay will represent a robust, fast, sensitive and precise immunoassay for the determination of anti-Müllerian hormone concentration.