Abstract: Cholesterol, phospholipid and fatty acid metabolism played an important role in occurrence of atherosclerosis and inflammation-cancer transformation. Quantitative analysis of many lipids in the three metabolism pathways in a single run was an enormous challenge, due to the huge differences in physicochemical properties and concentration levels of pre-selected metabolites in biofluid samples. Based on this, the serum sample was extracted ultrasonically in the mixture of methanol and dichloromethane at a volume ratio of 65∶35, and then 145 lipid components in three metabolic pathways were determined simultaneously by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) within 40 min, with lower limits of determination in the range of 0.03-50.0 μg·L^-1. Internal standards of phosphatidylglycerol(15:0)PG(15:0), 1-heptadecayl-glycerol-3-phosphorylcholineLysoPC(17:0), 7α-hydroxy-4-cholesterol-3-one-D7, ¹³C-glycylcholic acid and d₈-arachidonic acid were the representative research objects to evaluate the matrix effect, precision and accuracy of this method. As shown by the results, the values of matrix effect factor were found in the range of 76.7%-115%, indicating that the matrix effect was weak. In the spiked recovery test, the recovery of the target was in the range of 71.3%-110%, with RSDs (n=5) of the determined values less than 10%. This method was applied for determination of 145 lipid components in the serum from 30 healthy people and 30 hepatitis B (HBV) patients, and the 29 differential metabolites screened were analyzed by thermography and correlation analysis. It was shown that the levels of phospholipid (PL) and conjugated bile acid (BA) were elevated, but the levels of lysophospholipid (LPL) and fatty acid (FA) were decreased in serum of HBV patients compared with healthy people. According to correlation analysis test of differential metabolites, there was a significant positive correlation between BA and PL expression, and there was a significant negative correlation between LPL and PL expression. Moreover, as shown by the analytical results from receiver operating characteristic (ROC) curve, the area under the ROC curve (AUC) value of LysoPA(16:0) was 1.000, which meaned that LysoPA(16:0) was a very potential biomarker for HBV diagnosis.