Objective To observe the effect of different storage time on 14 blood biochemical indexes in rats. Methods Randomly selected 40 adult SD rats were included in this study. Fasting venous blood samples were collected, serum was separated, sealed, and stored in the refrigerator (4℃ and -20℃). The serum parameters were detected at 0 h,4 h,24 h,96 h and 7 d, respectively, using an automatic biochemical analyzer.A total of 14 blood biochemical indexes were detected, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), creatinine (CREA-J), uric acid (UA), urea nitrogen (UREA), blood glucose (GLU), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), creatine kinase (CK) and lactate dehydrogenase (LDH). The effects of serum storage time on blood biochemical results were compared. Results The trends of blood biochemical data in male and female rats were consistent. Compared with the indexes of serum preserved at 4℃ for 0 h, the ALP was significantly reduced after storage for 4 h, 24 h, 96 h, and 7 d (P< 0.05), ALB were significantly increased after 96 h and 7 d (P< 0.01), CREA-J was significantly increased after 96 h, 7 d (P< 0.05), UA was significantly increased after 24 h, 9 h, and 7 d (P< 0.01), and no significant changes in other indicators (P> 0.05). Compared with the values of 0 h serum, the serum preserved at -20℃ showed that ALT was significantly increased after 7 d (P < 0.01), AST significantly increased after 96 h and 7 d (P< 0.05), TP significantly decreased after 4 h and 24 h (P< 0.05), ALB significantly increased after 4 h, 24 h, 96 h, and 7 d (P< 0.01), CREA-J significantly increased after 24 h, 96 h, and 7 d (P< 0.01), UA significantly increased after 4 h, 24 h, 96 h, and 7 d (P< 0.01), TC significantly increased after 4 h, 24 h, 96 h, and 7 d (P< 0.01), TG significantly increased after 96 h and 7 d (P< 0.05), CK significantly increased after 96 h and 7 d (P< 0.05), LDH significantly increased after 96 h and 7 d (P< 0.05), and no significant changes in other indicators (P> 0.05). Conclusions The biochemical tests of rat serum should be immediately performed as they were collected, especially for ALP test. For the sera stored at 4℃, the test should be finished in different times:UA test in 4 hours, ALB and CREA-J test in 24 hours, and ALT, AST, TP, UREA, GLU, TC, TG, LDL-C, CK, and LDH test in 7 days.