Osteoarthritis is a prevalent aging disease in the world, and in recent years it has shown a trend toward younger age, which is becoming a major health problem in the world and seriously endangers the health of the elderly. However, the etiology and pathogenesis of osteoarthritis are still unclear, causing great trouble for treatment. To screen out candidate biomarkers that could be used for the identification of osteoarthritis and explore the pathogenesis of osteoarthritis, we performed an untargeted metabolomics analysis of nine New Zealand rabbit serum samples by LC-MS/MS, including three normal serum samples (control group) and six osteoarthritis serum samples (case group). Finally, 44 differential metabolites were identified, and the ROC analysis results indicated that a total of 36 differential metabolites could be used as candidate biomarkers. Further metabolic pathway enrichment analysis was performed on these differential metabolites, and we found that a total of 17 metabolic pathways were affected, which may provide directions for the study of osteoarthritis mechanisms.