Atherosclerosis was a chronic inflammatory disease with activation of both innate and adaptive immunity. Lipids, especially galactosylceramides were recognized as antigens and stimulator of natural killer T (NKT) cells and produced cytokines release. In this work, we established a novel online normal- and reversed-phase two dimensional (2D) liquid chromatography (LC) quadrupole time-of-flight mass spectrometry (QToF-MS) method for lipidomic analysis. This method was applied to identify the lipid products in plasma and making its merits in quality and quantity of the detection of lipids. The differences of lipids metabolism products in human plasma between carotid atherosclerosis patients and control subjects were identified in details. 518 endogenous lipid species from 17 classes were determined in human plasma. And we successfully achieved chromatographic separation of galactosylceramides (GalC) and glucosylceramides (GluC), which are isomers and have the same molecular weight. Only the levels of GalC in atherosclerosis patients were significantly increasing, rather than GluC, compared with the controls (controls vs. patients: the ratio was 1.5-2.8 fold increasing, P＜0.05). The increasing levels of GalC in atherosclerosis groups were revealed that immune responses were important of atherosclerosis exiting and progression. It would be helpful to the well understanding atherosclerosis and further investigation.