The aim of this study was to compare the bioequivalence and safety of test preparation sodium levofolinate injection with reference preparations of calcium levofolinate for injection and sodium folinate for injection in China. A singlecenter, randomized, open-label, 3-period, crossover test was conducted on 24 healthy subjects. Plasma concentration of levofolinate, dextrofolinate, and their metabolites L-5-methyltetrahydrofolate and D-5-methyltetrahydrofolate were quantified by a validated chiral-liquid chromatography-tandem mass spectrometry method. All adverse events (AEs) were documented to evaluate safety as they occurred and evaluated descriptively. Pharmacokinetic parameters (maximum plasma concentration, time to maximum concentration, area under the plasma concentration-time curve over the dosing interval, area under the plasma concentration-time curve from time 0 to infinity, terminal elimination half-life, and terminal rate constant) of 3 preparations were calculated. A total of 8 subjects (10 cases) of AEs occurred in this trial. No serious AEs or unexpected serious adverse reactions were observed. Sodium levofolinate was bioequivalent to calcium levofolinate and sodium folinate in Chinese subjects, and the 3 preparations were all well tolerated.