To explore the possibility of photodisruption in rabbit sclera by femtosecond (fs) laser and seek appropriate incision techniques and relevant parameters, a fs laser (800 nm/50 fs) with different pulse energies was applied to irradiate rabbit sclera in vitro. By moving a computer-controlled three-axis translation stage to which the sample was attached, the laser achieved three types of incisions: transscleral channel, snake pattern and linear cut. The irradiated samples were observed by light microscopy and scanning electron microscopy (SEM). In comparison with fs laser, Nd:YAG was used as control. The experimental results show that through an objective lens with numerical aperture (NA) of 0.2, the fs laser with power intensity larger than 955 TW/cm2 and pulse energy ranging from 37.5-125 mJ, cutting depths from 30-70 mm may be achieved after linearly scanning in sclera at a translation speed of 0.1 mm/s. However, it failed to make any photodisruption when the power intensity was below 796 TW/cm2 or the pulse energy was less than 31.25 mJ under the same condition. Compared with the Nd:YAG laser, the inner wall of the channel was smoother and the damage to the surrounding tissues was slight with the fs laser. The high precision of intrascleral photodisruption and minimal damage to surrounding tissues by a fs laser display its potential application in the treatment of glaucoma.