Objective To visualize the process of introcular flow caused by injection into the posterior chamber of the impermeable vitro eyeball with particle image velocimetry (PIV) technology, and calculate the flow fields at different moments, so as to investigate PIV experimental scheme for low-speed flow field measurement in the eye and provide basis for the in vivo measurement of aqueous humor flow under physiological status. Methods In an impermeable vitro eyeball, the introcular flow would be slow enough when the injection pump was driven at the rate of 0.2, 0.4, 0.6, 0.8, 1.0, 1.5 mL/min. Fluorescent particle solution, with a certain concentration and particle diameter of 10 μm, was injected into the posterior chamber of the vitro rabbit eye, and the sheet laser was projected to the medial frontal plane of the eye. Then clear particle images were captured by camera, and the velocity field was recorded and calculated by PIV system. Results The fluid into the posterior chamber first filled in the posterior chamber and the pupil, then passed the pupillary margin and flowed into the anterior chamber, which was consistent with the theoretical flow process of aqueous humor under physiological conditions. Based on analysis of the particle images, the velocity inside an impermeable eyeball was calculated at the magnitude of a few millimeters per second. Conclusions PIV method can be applied to low-speed flow field measurement, and the flow characteristics inside the eyeball can also be measured by PIV method, which contributes to the measurement of aqueous flow under both physical and pathological conditions, provides experimental verification for numerical simulations on aqueous humor field, and offers a new diagnostic and treatment perspective for shear force damage and destructions of corneal endothelial cells, the iris and lens under different flow fields.