A comprehensive strategy for quality evaluation of Atractylodis macrocephalae rhizoma by combining quantitative analysis of multi-components by single marker and HPLC fingerprint qualitative analysis was developed and validated in this paper. By analyzing chromatograms of 18 batches of Atractylodis macrocephalae rhizoma, the reference fingerprint of Atractylodis macrocephalae rhizoma was generated and 10 common peaks were identified, of which Atractylenolide I, atractylenolide II, atractylenolide III and atractylone were identified with chemical references. With atractylenolide III as an internal reference substance, the contents of the other three components in 18 batches of Atractylodis macrocephalae rhizoma samples were simultaneously determined by quantitative analysis of multi-components by single marker which were not significantly different from the results determined by external standard method (t test, P>0.839). The accuracy, precision, reproducibility and stability of this method were validated which exhibited satisfactory results, indicating that quantitative analysis of multi-components by single marker could be used for quantitative analysis of Atractylodis macrocephalae rhizoma instead of external standard method. The content of each component in 18 batches of Atractylodis macrocephalae rhizoma was significantly different from each other. There is no Assay specified in the quality standard of Atractylodis macrocephalae rhizoma in Chinese Pharmacopoeia (volume I) (2020 edition). This method combining quantitative analysis of multi-components by single marker and HPLC fingerprint can evaluate quality of Atractylodis macrocephalae rhizoma samples more comprehensively which is beneficial to the application of Atractylodis macrocephalae rhizoma.