Objective To study the effects of experiment-related factors on hematological parameters in SD rats, analyze the data difference and causes, understand the effects of anesthetics and stress responses on the physiological aspects of animals, and to provide a reference for the standardization of animal welfare and compound toxicity testing methods. Methods According to gender (A), fasting time (B), anesthesia (C) and blood collection mode (D), SPF SD rats were divided into 24 groups. Blood samples were collected from each group. Then, red blood cell count, hemoglobin levels, white blood cell count and classification indicators were measured. Results The primary and secondary order of the factors affecting the white blood cell count was D > C > A > B, and the levels of white blood cell count of each factor were male rats > female rats, and venous blood > arterial blood, chloral hydrate > pentobarbital sodium > no anesthesia. The primary and secondary order of the factors affecting the white blood cell classification was C > D=A=B, and factors affecting the levels of white blood cell classification were chloral hydrate > pentobarbital sodium > no anesthesia. The primary and secondary order of the effects of the factors on the red blood cell count and hemoglobin level was C > D=A=B, and the levels of red blood cell count and hemoglobin level were pentobarbital sodium > chloral hydrate> no anesthesia. There was no significant difference in the blood indexes between the different fasting time groups. Conclusions There is no effect of fasting on hematological parameters, but there are differences in the blood parameters between arteries and veins. The effect of chloral hydrate anesthesia on the count and classification of white blood cells is greater than that of pentobarbital sodium. The effect of chloral hydrate anesthesia on the red blood cell count and hemoglobin level is greater than that of pentobarbital sodium. The two kinds of anesthesia methods have their own advantages and disadvantages.