Clonorchiasis, an infectious disease caused by the liver fluke Clonorchis sinensis, may lead to the development of liver and gallbladder diseases, and even cholangiocarcinoma (CCA). However, the pathogenesis, host-pathogen interaction, and diagnostic markers for clonorchiasis remain unclear.Eighteen rabbits were randomly divided into control group (n = 9) and C. sinensis-infected group (n = 9), and their plasma samples were collected at 7, 14, 28, and 63 days post-infection (dpi). Biochemical indices and metabolites in different infection periods were detected. A non-targeted ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach was employed to investigate the metabolic profiles of plasma in rabbits, and related metabolic pathways of differential metabolites and correlation between candidate biochemical indices and differential metabolites were analyzed. Finally, the candidate biomarkers were verified with human samples using a targeted metabolomics method.The result of biochemical indices indicated C. sinensis infection would affect the liver function biochemical indices, especially alanine aminotransferase, aspartate transaminase (AST), glutamyl transpeptidase (GGT), total bile acid, high-density lipoprotein, and cholinesterase. The metabonomic results showed that 58, 212, 23, and 21 differential metabolites were identified in different phases of the infection. Multivariate statistical analysis of differential metabolites revealed distinct metabolic signatures during different phases of infection, with most of these signatures being observed at 14 dpi, which mainly influences the amino acid metabolisms. For metabolites and biochemical indices, AST, GGT, hypoxanthine, L-pipecolic acid, and D-glucuronate represented potential noninvasive biomarkers for the diagnosis of C. sinensis (P < 0.05 and AUC > 0.8). Furthermore, GGT and D-glucuronate levels were positively correlated with the infection (r(28) = 0.98, P < 0.0001) and showed excellent diagnostic performance (AUC = 0.972; 95% confidence interval, 0.921 to 1.000).The present results provide new insights into plasma metabolic changes in rabbits during C. sinensis infection, and the potential biomarker may be used for developing an effective method to diagnose clonorchiasis in the future.© 2022. The Author(s).