Abstract Objectives The aim of this study was to establish reference intervals (RIs) of homocysteine (Hcy) in healthy Chinese Han ethnic adults according to the Clinical and Laboratory Standard Institute (CLSI) CA28-A3. Methods After filtering, serum Hcy values in 20,810 healthy subjects from a middle area of China (Wuhu, Anhui province) were measured. The non-parametrical percentile method was used to establish RIs and the 90% confidence intervals of lower and upper limits were calculated. The relationship between Hcy and age was analyzed by using Spearman’s approach. Besides, the risk of HHcy in males and females was examined by logistic regression analysis. Results The RIs of Hcy were 9.10–20.20 μmol/L for males, 6.10–15.90 μmol/L for females and 8.00–19.80 μmol/L for total subjects from 20 to 90 years old. The serum Hcy level was significantly correlated with age both in males (r=0.2159, p<0.0001) and females (r=0.2955, p<0.0001). In males, the prevalence and the risk of HHcy were higher than females of all ages (p<0.001). Conclusions Through the analysis of a large dataset from healthy population, it showed that the variations in different age- and sex-related RIs of Hcy were significant. It suggested that establishing more specific age- and sex-related RIs for Hcy in China is necessary.