Objective To develop a high accuracy method for non-invasive intratracheal instillation in rats. Methods The plastic catheter of disposable venous infusion needle was cut into a cannula,which was then intubated by transillumination.A microsyringe was introduced into the rat trachea through the cannula before the intratracheal instillation of saline solution was performed.The instilled volume ranged from 5μl to 200μl and the accuracy is 1μl. Results The intratracheal instillation operation was performed successfully for 50 times in 10 rats weighing 200~350g.The general health status was not affected during or after the operation in the rats.Conclusion The method developed in our study for intratracheal instillation is characterized by non-invasion, accuracy and safety,which could be widely used in intratracheal drug administration or poisoning in rats.