Introduction Acute-on-chronic liver failure (ACLF) is a fatal syndrome that presents with acute deterioration of liver function in chronic hepatitis B virus (HBV) patients. However, reliable diagnostic and prognostic biomarkers are scarce. Objectives The aim of this study to identify lipid species associated with HBV infection as well as novel lipid biomarkers for HBV-ACLF. Methods High performance liquid chromatography-tandem mass spectrometry was used for targeted lipidomic analyses of 147 lipid species. Fasting-state plasma samples from 74 HBV-ACLF patients, 86 HBV-non-ACLF patients [ 30 HBV-immune tolerant (HBV-IT) and 56 chronic hepatitis B] and 20 healthy controls. Univariate and multivariate analyses examined changes in lipid species among patient groups. Results The HBV-ACLF and HBV-non-ACLF groups had distinctly different lipid profiles, while the HC and HBV-IT groups had similar lipid profiles. Further, lysophosphatidylcholine (LPC) 22: 6, cholesterol ester (CE) 22: 6, CE 20: 4, CE 18: 2 and CE 18: 1 could be used as potential biomarkers for the early prediction of ACLF. Meanwhile, univariate and multivariate analyses identified CE 20: 4, LPC 16: 0, LPC 18: 0, phosphatidylcholine (PC) 40: 6 and PC 32: 0 as putative diagnostic biomarkers of HBV-ACLF. Moreover, LPC 16: 0 and LPC 18: 0 were significantly associated with model for end stage liver disease (MELD) scores, and the two lipid species combined with MELD score had significant capability to predict the 6-month mortality. Conclusions Our study revealed that lipid metabolism disorders were significantly associated with the severity of liver inflammatory injury rather than HBV infection in patients with chronic HBV infection, and specific lipid species could be used as potentially biomarkers for diagnosis and prognosis in HBV-ACLF.