BACKGROUND: A total of 300 SD rats were used for blood sampling. In abdominal aorta approach, the best puncture point was the abdominal aortic bifurcation 1-3 mm towards the heart, with a success rate of 93.6%. In posterior orbital venous plexus approach, the needle was vertically inserted into the inner canthus and rotated toward the eyeground to open venous plexus (success rate 89.9%). In cardiac puncture approach, below the xiphoid process, the needle punctured into the skin with 25°-30° oblique upward, through the diaphragm until 2.5-3.0 cm deep (success rate 83.4%). In tail end approach, surgical scissors cut off 5-10 mm tail top (success rate 94.4%). In jugular vein approach, the needle was horizontally inserted along the fourth rib into the skin until the jugular vein, about 5 mm deep, at 30°-40° with the chest surface (success rate 80.9%). A large blood volume could be obtained by abdominal aorta approach, which leads to less haemolysis and no hurt to organs, no gas embolism or haemostasis caused by inappropriate operation. But each approach has advantages and drawbacks, the selective principle should be based on experimental require.