Extracellular vesicles (EVs) consist of lipid bilayers, occur in various biofluids, and are invaluable in biomarker screening. Liquid chromatography coupled with high-resolution mass spectrometry (LC-MS) was recently used to study comprehensive EV lipid profiles in vitro. The aim of this study was to establish a lipidomics platform for human plasma and serum EVs for comprehensive characterization of their lipid profiles, and to compare them with those of other lipid-containing particles, such as high-density lipoproteins (HDL), and low/very low-density lipoproteins (LDL/VLDL). Isolation was validated by specific protein markers; CD9 and MHC class for EVs, apoA-I for HDL, and apoB-100 for LDL/VLDL. Lipidomics identified 264 lipids from isolated plasma EVs, HDL, and LDL/VLDL. The absolute lipid levels per unit protein content in the EVs were more than eight times lower than those of the lipoproteins. Moreover, the EVs had higher lysoglycerophospholipid levels than HDL or LDL/VLDL. Similar profiles were also determined for human serum. The present study found that the lipid profiles of EVs are unique and distinctly different from those of lipoproteins. The lipidomics platform applied to human plasma and serum EVs could generate important information for the exploration and qualification of biomarkers in disease diagnosis.