To identify serum biomarker(s) for predicting knee cartilage volume loss over time, we studied 139 knee osteoarthritis (OA) patients from a previous 24-month clinical trial cohort. Targeted metabolomic profiling was performed on serum collected at baseline. The pairwise metabolite ratios as proxies for enzymatic reaction were calculated and used in the analysis. Cartilage volume loss between baseline and 24 months was assessed quantitatively by magnetic resonance imaging (MRI). Data revealed an association between the serum ratio of lysophosphatidylcholine 18:2 (lysoPC 18:2) to phosphatidylcholine 44:3 (PC44:3) and the cartilage volume loss in the lateral compartment (β = −0.21 ± 0.04, p = 8.53*10⁻⁷) and with joint degradation markers, COMP (r = 0.32, p = 0.0002) and MMP1 (r = 0.26, p = 0.002). The significance remained after adjustment for age, sex, BMI, diabetes, hypertension, dyslipidemia, and the treatment taken in the original study. As the ratio indicated the over activation of the conversion pathway of PC to lysoPC catalyzed by phospholipase A₂ (PLA₂), we assessed and found that a specific PLA₂, PLA₂G5, was significantly increased in human OA cartilage and synovial membrane (85% and 19% respectively, both p < 0.04) compared to controls, and its overexpression correlated with IL-6 (r = 0.63, p = 0.0008). Our data suggest that the serum lysoPC 18:2 to PC44:3 ratio is highly associated with a greater risk of cartilage volume loss of the knee and warrants further investigation in an independent cohort.