Abstract: L-ergothioneine is a sulfur-containing histidine derivative, which has strong antioxidant, anti-aging and sunscreen effects, and has been widely used in nutrition, cosmetics and other fields. However, there is no research on relevant standard substances in China. To ensure the traceability, comparability, and accuracy of L-ergothioneine testing in cosmetics, a purity-certified reference material for L-ergothioneine has been developed. The raw materials' purity is evaluated by employing both mass balance approach and quantitative NMR (nuclear magnetic resonance) spectroscopy techniques. The standard value is established based on the average of the results obtained from these two analytical techniques. In the mass balance method, the principal component structure analogues were quantitatively determined by liquid chromatography, the water was determined by Carl Fischer method, the non-volatile impurities were determined by inductively coupled plasma mass spectrometry, the residual solvent was determined by headspace gas chromatography. The internal standard of quantitative nuclear magnetic method adopts the national certified standard material GBW06151 Acesulfame purity standard material. The uncertainty of the reference material is evaluated by considering the contributions from the fixed value, uniformity, and stability. The determined purity level of the L-ergothioneine reference standard is 99.6%, with a relative extended uncertainty of 0.2% (k=2). The reference material can be used for ergothioneine detection in food, cosmetics and other fields, quality control in cosmetics production departments and related testing laboratories, and analysis method confirmation and evaluation.