In order to explore the possibility of photodisruption in rabbit sclera by femtosecond laser and to seek the appropriate ways of incision and relevant parameters, femtosecond laser (800 nm/50 fs) with different pulse energies was applied to irritate rabbit sclera in vitro. By moving a three-axis, computer-controlled translation stage to which the sample was attached, femtosecond laser could achieve three types of incisions, including transscleral channel, snake pattern and linear cutting. The irritated sclera was observed by light microscopy and scanning electron microscopy (SEM). In comparison with femtosecond laser, Nd:YAG laser was used as control. It was shown that through a 0.2 numerical aperture (NA) objective lens, femtosecond laser with the power intensity larger than 9.55×1014 W/cm2 and the pulse energies ranging from 37.5 to 125 μJ could achieve cuttings with the depth from 30 to 70 μm after linearly scanning on the sclera at the speed of 0.1 mm/s. Whereas, it failed to make any photodisruption if laser power intensity was below 7.96×1014 W/cm2 or the pulse energy was less than 31.25 μJ under the same condition. Comparing with Nd:YAG laser, the inner wall of channel was smoother and the damage to surrounding tissues was slighter by femtosecond laser. The high precision and minimal damage to surrounding tissues with femtosecond laser predicted its potential use in the treatment of glaucoma.