Objective Beagle dogs are commonly used animal for drug safety evaluation. As the necessary parameters, blood biochemical indicators are detected in acute or chronic toxicity tests. This study aims at assessing the influence of different preservation conditions and different preservation time on blood biochemical indicators to ensure the reliability of test results of long-term toxicity assessment.Methods Six Beagle dogs (3 males and 3 females) were used in this study. After collection and preparation of serum samples, biochemical indicators were detected after preservation in refrigerator at 2-8℃ for 1, 2, 5, 8, and 12 hours; after preservation in ice transportation boxes at 2-10℃ for 2, 5, and 8 hours; and after preservation in refrigerator at-20℃ for 1, 3, and 5 days. The biochemical indicators included alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), total protein (TP), albumin (propagated), urea, creatinine (CREA), glucose (GLU), total cholesterol (TCHO), total bilirubin (TBIL), creatine kinase (CK), gamma pancreatic acyl transferase (GGT), calcium (CA), lactate dehydrogenase (LDH), phosphorus (P), high-density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglyceride (TG), sodium (Na⁺), potassium (K⁺) and chloride (Cl^-). Results Compared with the results of samples preserved for 1 hour, the LDL-C result of that preserved in refrigerator at-20℃ for 5 days was significantly increased (P<0.05), and the coefficient of variation was 39%. The CK and LDH results of specimens preserved in refrigerator at-20℃ for 5 days was increased but not significantly (P>0.05), and the coefficient of variation of LDH was 41%. Conclusions According to the test results of blood biochemical indicators in the Beagle dogs detected after different preservation conditions and different preservation time in this study, detection test should be done within 1 hour, if not, detection should be done within 12 hours for the samples preserved at 2~8℃, or within 3 days for the sample preserved at-20℃. For transportation of serum samples, the serum samples should be placed in the ice box at 2~10℃, and detection test should be done within 8 hours.