Abstract: Vitamin D deficiency and insufficiency is a global health issue that afflicts people of different age groups all over the world. As a reliable indicator for the evaluation of vitamin D, 25OHD is usually determined by LC-MS/MS. The tandem mass spectrometry based on ion trap has the characteristics of simple structure and high sensitivity. However, there are some problems such as narrow linear range and space charge effect, which limit its analysis of trace substances in complex matrix samples. In this study, the device and technology of a new quadrupole linear ion trap mass spectrometry was used to reduce the space charge interference of trace or ultra trace target ions by high abundance impurity ions in complex matrices. For the first time, isotope dilution mass spectrometry for accurate measurement of serum 25OHD was established by QLIT-6610MD, which is quadrupole-linear ion trap tandem mass spectrometer, and was independently developed by Technology Innovation Center of Mass Spectrometry for State Market Regulation, Center for Advanced Measurement Science, National Institute of Metrology. Serum samples were precipitated with methanol, extracted with n-hexane, dried with liquid nitrogen, redissolved in the initial mobile phase, and determined by QLIT-6610MD. The matrix-matched standard curve was used for quantification. The linear ranges of 25OHD₂ and 25OHD₃ were in the range of 0.78-50 μg/L and 1.56-100 μg/L with R²>0.999 9, respectively. The detection and quantification limits of 25OHD₂ and 25OHD₃ were 0.06, 0.12 μg/L and 0.02, 0.06 μg/L, respectively. The precision of quality control was better than 4.06%. The recoveries ranged from 99.25% to 108.38%, and the coefficients of variation were all less than 5.1%. After the method confirmation, the quantitative results of clinical samples were compared with triple quadrupole tandem mass spectrometry. 50 Clinical serum samples were measured and compared by QLIT-6610MD, AB 4500MD and YS EXACT 9900MD. The correlation and consistency of the data were analyzed by Pearson and Bland-Altman. The correlation coefficient of the three groups data was R≥0.998 0, P<0.01, and the results of Bland-Altman analysis showed good consistency, indicating that the results measured by the three instruments were consistent, which meant that QLIT-6610MD can meet the needs of clinical diagnosis. QLIT-6610MD achieved the same level of triple quadrupole mass spectrometry to determine the concentration of 25OHD in serum. It can not only become a new choice for clinical mass spectrometry, but also lay a foundation for the development of domestic mass spectrometer. Based on the direct axial coupling technology of quadrupole and linear ion trap, QLIT-6610MD can greatly reduce the interference of matrix ions and make the ion trap detect target ions more effectively. This technology has a strong potential to further improve the measurement accuracy of trace or ultra trace targets in complex matrices. In the future, it can be applied to mass spectrometry monitoring scenarios that needs to meet the requirements of high accuracy, high sensitivity, on-site speed and low cost at the same time.