Objective To establish an accurate and sensitive method for determination of Vitamin K₁ in human serum by liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods The stable isotope, ₂H₇-vitamin K₁ was employed as internal standard for analysis on AB SCIEX API 4000. The serum samples were prepared by precipitating proteins by methanol, and further liquid-liquid extracted by n-Hexane. Then the residuals were re-dissolved in acetonitrile and analyzed by LC-MS/MS in the positive ion modewith multiple reaction monitor (MRM) pattern by electrospray ionization (ESI)．Results The LLOQ of the established method was 0.1 ng/mL. The linearity of vitamin K₁ was well (r=0.9992) in the range of 0.1~20 ng/mL. The relative standard deviations (RSDs) of intra- and inter-day precisions of low and high concentration quality control samples were less than 15%.The average recoveries were in the range of 92.83~99.32%. Conclusions In this study, a rapid and accurate isotope dilution LC-MS/MS method was developed for determination of Vitamin K₁ in the human serum, which can be used as a routine inspection for the clinical analysis of vitamin K₁.