Abstract: Objective To compare the differences in serum lipid profiles between patients with biliary obstruction and healthy individuals. Methods A total of 55 patients with biliary obstruction who were admitted to the Affiliated Hospital of Xuzhou Medical University from March to June 2023 were enrolled, while 25 healthy individuals were set as controls. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was applied to detect serum lipid metabolites. Then, differentially expressed lipid metabolites were screened by principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), fold change (FC), t-test, and variable importance in projection (VIP) values. Lipid metabolic pathway analysis was performed using the MetaboAnalyst platform. Results A total of 31 differential lipids were identified, including 3 ceramides (Cer), 4 cholesteryl esters (CE), 5 phosphatidylethanolamines (PE), 3 lysophosphatidylcholines (LPC), 9 phosphatidylcholines (PC), 6 sphingomyelins (SM), and 1 sterol (ST). Pathway enrichment analysis revealed that the two most significantly enriched pathways were glycerophospholipid metabolism and sphingolipid metabolism. Conclusions Patients with biliary obstruction exhibit distinct alterations in serum lipid profiles compared to healthy individuals, particularly involving Cer, CE, PE, PC, and LPC.