Objective To improve the orotracheal intubation verifying technique and reduce the complication of backflow in rat experiment. Methods A new position evaluation of anti-backflow device was designed and made of safety IV catheter and closed IV catheter system. 60 adult male Sprague Dawley rats 216±20 g were randomly assigned to two groups:group A (n=40) for verifying placement, group B (n=20) for anti-backflow test. Group A was further divided into group A1 using self-designed positioning device, group A2 using aerosol, group A3 taking cotton fiber for positioning judgment. The group B was divided into two subgroups, B1 and B2, counting escaped bubbles as a means of positioning observation, the difference is that group B1 using frustum of a cone shape anti-backflow device, while the group B2 using common airway tube. Routine endotracheal intubation was performed to observe and record the time of positioning, the location of exhalation phase, and the length of inspiratory phase countercurrent water column. The group A1 further performed tracheotomy under direct vision clearly to confirm the anatomic positioning status. Results During the exhalation cycle,three or more bubbles were observed to escape continuously, indicating that the intubation tube was properly placed and open in the airway. Positioning time:It took 1.75±1.02 respiratory cycles in group A1,3.30±0.95 respiratory cycles in group A2 and 4.10±0.99 respiratory cycles in group A3 to complete the assessment the positioning status. There was no statistically significant difference between groups A2 and A3 (P>0.05). The time needed for group A1 was significantly shorter than that of groups A2 and A3 (P<0.01). The longest countercurrent water column length in group B1 was 3.23±0.53 cm, and 8.48±1.01 cm in the group B2. Conclusions The new designed anti-backflow positioning evaluation device is a simple and convenient appliance to evaluate the location of orotracheal intubation in rat experiment. It can effectively improve the positioning efficiency and has practical application value.