Abstract: The demand for accurate mass spectrometry measurement in the field of clinical diagnosis is increasing. In the face of foreign monopoly, our team has developed new technologies and new methods for the selective enrichment of trace targets in complex matrices by ion traps, and has developed a quadrupole-linear ion trap (quadrupole-linear ion trap, Q-LIT) tandem mass spectrometer with independent intellectual property rights and a liquid chromatography tandem mass spectrometry system (model QLIT-6610MD), which have obtained a medical device registration certificate in China. In this study, a home-built Q-LIT liquid chromatography-tandem mass spectrometer QLIT-6610MD was developed to measure vancomycin in serum with high accuracy . After protein precipitation and liquid-liquid extraction, the samples were analyzed by QLIT-6610MD. The endogenous substances in the samples did not interfere with the quantification. The results showed that vancomycin has a good linear relationship of signal intensity vs. concentration in the range of 1.56-50.0 mg/L with the linear correlation coefficient (R²)≥0.99, and the limit of detection and limit of quantitation are 0.19 and 1.56 mg/L, respectively. Intra-day and inter-day precisions are 1.62%-3.82% and 2.07%-5.07%, respectively. The recoveries of vancomycin in serum samples are 95.97%-97.74%. The coefficient of variability (CV) of matrix effect normalization factor is less than 7.60%, CV of sample stability is better than 4.26%, and CV of long interval sample analysis is better than 2.13%. The method was verified by the determination of low, medium and high quality controls, and the results were all within the range of target values. QLIT-6610MD, AB API4000 and YS EXACT 9900MD were used to determine 42 real clinical serum samples. The correlation and consistency of the data were analyzed by Deming regression, Bland-Altman analysis and Pearson correlation analysis. The results showed good consistency among the results obtained by the three spectrometers, indicating that the data of three instruments are comparable, which prove that the home-made QLIT-6610MD can reach the quantitative accuracy of triple quadrupole-mass spectrometer. The instrument can effectively reduce the interference of matrix ions and enhance the capture efficiency of target ions, based on quadrupole-linear ion trap tandem mass spectrometric technique. QLIT-6610MD has great potential to provide a new instrumentation solution for real-time clinical analysis in the future.