Background It has been widely reported that the key biomarkers of homocysteine metabolic pathways have emerged as markers or independent risk factors for cardiovascular disease and many other diseases. To improve the accuracy of evaluation in China, we establish the reference intervals of serum homocysteine (Hcy), cysteine (Cys), and methionine (Met) in apparently healthy Chinese adults. Methods We examined 1553 subjects aged ≥20 years who underwent Hcy metabolic pathway tests, in the Chaoyang Central Hospital between January 2019 and December 2020. The serum Hcy, Cys, and Met levels were simultaneously determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Multiple regression analysis was performed to identify sources of variation. A nonparametric method was used to establish the reference intervals. Results Gender differences were observed in Hcy and Met, with higher levels in males. Reference intervals for males were 6.09–17.00 μmol/L (Hcy) and 19.31–33.68 μmol/L (Met); conversely, the reference intervals for females were 4.61–14.61 μmol/L (Hcy) and 16.16–30.35 μmol/L (Met). The serum Cys reference intervals were seen to be gender-independent, but age-dependent reference intervals were needed; those for Cys were 188.85–334.27 μmol/L, 200.48–340.66 μmol/L, and 216.03–349.67 μmol/L in <40, 40–50, and ≥50 years, respectively. Conclusions By this study, the gender- or age-specific reference intervals of Hcy, Cys, and Met that were matched with the Chinese population were established, providing valuable references for clinical work and laboratory researches.