Background: Wistar rats are extensively used as the model for assessing toxicity and efficacy in preclinical research.Hematological and biochemical laboratory data are essential for evaluating specific variations in the physiological and functional profile of a laboratory animal. Establishing hematological and biochemical reference values for Wistar (han) rats at various age intervals was the goal of this work. Male and female Wistar rats (n = 660) of ages 6–8 weeks, 10–14 weeks and > 6 months were used in the experiment. Blood and serum were collected from these rats under fasting conditions. Results: We observed that the majority of hematological and biochemical parameters were significantly influenced by sex and age. Hematological changes were significantly correlated to aging were increased red blood cells, hemoglobin, hematocrit, neutrophils, monocytes and eosinophils in both sexes, as well as decreased platelet, mean corpuscular volume, mean corpuscular hemoglobin and lymphocytes in both sexes. White blood cells of male rats were considerably higher than those of female rats in all age ranges. For biochemistry, increase in glucose, total protein and creatinine were seen in both sexes, along with increases in urea in females and alanine aminotransferase in males.Age was significantly associated with decreased alkaline phosphatase in both sexes. Conclusions When using Wistar rats as a model, these reference values may be useful in evaluating the results.