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Objective To find a proper method of puncturing into the posterior chamber of the rabbit eye and to measure the pressure in posterior chamber and the pressure difference between anterior and posterior chambers in vivo. Methods With the help of the high-accuracy pressure and pressure difference sensors, the pressure in posterior chamber and the pressure difference between anterior and posterior chambers were measured continuously in normal rabbit eyes with anesthesia in vivo. The needle directly punctured into the posterior chamber at 1 to 1.5 millimeters away from the cornea-sclera limbus, penetrated the sclera, and then slided horizontally the needle underneath the iris. Results The pressure value in posterior chamber was from 839.93Pa to 2662.48Pa, while the pressure difference between anterior and posterior chambers was from 46.15Pa to 85.52Pa with its average value 59.73Pa and the wave period was about 11.17min. Conclusions The puncture method for surveying the pressure of rabbit’s chambers does less hurt to the eye-ball and the experimental data of the pressures in posterior chamber and pressure difference between anterior and posterior chambers, are authentic and reasonable. This paper also provides a new method of in vivo detecting the pressure difference between anterior and posterior chambers for glaucoma patients.