Objective To introduce the operation skills of tail vein injection in conscious rats, and improve the success rate of ingection. Methods The rat was fixed by the operator with a self-made binding clothes, one person assists to fix the rat tail, one person performs puncture, and one person performs injection. During the injection process, we should minimize the injury to the rat tail and the stress caused by operation, strictly limit the needle point, the number of puncture, the maximum dose and injection speed, and to make the needle position away from the injection site to avoid contamination and waste of the drug solution. Results The operation method was successfully established and it was fast, stable, with good repeatability and high degree of coordination. Conclusion This operation is rapid, reliable and stable, worthy of recommendation, especially for the intravenous injection of expensive drugs.