A set of device for the in vivo measurement of the pressure difference between the anterior and the posterior chambers (PDAP) was designed to investigate the temporal varying rules of PDAP in the anterior segment of rabbit eyes. A platform was established for the in vivo measurement of PDPA according to the mechanism of joint implement. Rabbit models with high intraocular pressure (IOP) were constructed by means of injecting Carbomer into anterior chamber to increase IOP. The in vivo 24 hours continuous measurements of PDAP were performed for normal rabbit eye and eye with high IOP. The developed device could sensitively response to the small pressure difference in eye. The pressure difference in the normal rabbit eye varied with time, and the variation range during a whole day was 5.84–96.84 Pa which reflected the existence of physiological rule. For the rabbit eye with high IOP, pressure in anterior chamber was higher than that in posterior chamber which was in consistence with the theory of self-adaptation adjustment. The present study indicates that the approaches and device designed in this paper can well implement the measurement of PDAP as well as the temporal varying rules of PDAP in the anterior segment during a whole day.