Deep vein thrombosis (DVT) has become an important factor in the global disease burden. In this study, the differential metabolites in urine were screened by untargeted metabolomics to find metabolic markers to compensate for the poor specificity and single defect of D-dimer, γ-butyl betaine (GBB), and L-carnitine (L-CN). Ultra-high performance liquid chromatography-mass spectrometry ( UPLC-MS / MS ) was used to verify the levels of GBB and L-CN in clinical and rat blood and urine at different stages of DVT, and the diagnostic titer and correlation analysis of GBB and L-CN with DVT were analyzed in an all-round manner. The levels of GBB and L-CN in plasma and urine were lower in patients with DVT and in Sprague-Dawley rats than in the control group (P < 0.05), and the trends of GBB and L-CN were similar in plasma and urine of humans and rats with DVT. In human plasma, the area under the curve (AUC) of GBB combined with D-dimer was 0.914 (P < 0.001) in the acute group and 0.895 (P < 0.001) for L-CN combined with D-dimer in the subacute group. In human urine, the AUC of L-CN combined with GBB in the subacute group was 0.855 (P < 0.001). Therefore, GBB and L-CN are closely related to the development and progression of DVT and can be used to screen and diagnose DVT at different phases. Moreover, GBB and L-CN expression levels are similar in humans and rats, providing precise indicators and animal models for the in-depth study of the DVT formation mechanism.