Two trials were conducted to compare the differences of blood biochemical parameters from different sites in dairy cows. In trial 1, 10 animals were randomly selected from 35 Holstein cows (DIM = 202 ± 38.7; milk yield = 24.5 ± 3.8 kg/d; parity = 2.9 ± 1.2). Blood samples were collected from jugular vein, subcutaneous abdomen vein (SAV) and coccygeal vein into heparinized tubes at approximately 2 h after feeding, respectively, to investigate the differences of biochemical parameters among 3 sites of vein. In trial 2, 18 early lactation cows (DIM = 46 ± 12.6; milk yield = 42.6 ± 15.3 kg/d; parity = 3.3 ± 1.9) were used to collect the blood samples from coccygeal artery and vein into heparinized tubes at approximately 4 h after feeding, respectively. No hemolysis was found in both trials. ANOVA for biochemical parameters were performed using PROC GLM of SAS, and regression analysis was conducted using CORR of SAS. No differences were found in concentrations of creatinine, total bilirubin, total protein, albumin, and globulin, and activities of alanine transaminase, aspartate aminotransferase, and alkaline phosphatase among the 3 sites of veins (P > 0.05). Nonesterified fatty acids (NEFA) was higher (P = 0.02) in SAV than in jugular and coccygeal vein with no difference between jugular and coccygeal veins. β-hydroxybutyric acid (BHBA) was higher in jugular than in coccygeal vein, with higher value in coccygeal vein than in SAV (P < 0.01). Glucose concentration was higher in jugular vein than in SAV (P = 0.02). No differences were found in most of biochemical parameters between coccygeal artery and vein (P > 0.05), with higher concentration of glucose, NEFA and BHBA in coccygeal artery than in coccygeal vein (P < 0.01). Concentrations of glucose, NEFA and BHBA in coccygeal artery was closely correlated with those in coccygeal vein (R² = 0.842, 0.989 and 0.901, respectively, P < 0.01). The results from this study indicate that some of biochemical parameters may be different from blood site to site. Attention should be paid to these differences when the blood samples are analyzed.