Objective To develop a blood collection method for rats that is easy to perform and can be used for repeated, high-volume blood collection, and a method of positioning blood collection points. Methods The optimal entry point was determined by the body surface characteristics of rats, and blood was collected using a syringe needle to pierce the submaxillary venous plexus and then slowly draw blood. Results When rats were not anesthetized, a single operator completed the blood collection operation in 77. 46 s on average, and the average blood collection volume was 0. 53 mL. Two operators completed the blood collection in 56. 28 s on average, and the average blood collection volume is 0. 59 mL. Under anesthesia, blood collection by a single operator required 28. 67 s on average, and the average blood collection volume was 0. 56 mL. Conclusions The success rate of submandibular plexus blood collection is high, trauma to the animal is minimal, the operation is simple, and the blood collection volume is large, which can be used as the preferred choice for multiple blood collections from large numbers of live animals in pharmacological and toxicological experiments.