To evaluate the feasibility of middle ear bone ablation in-vivo with pulse CO 2 laser. Healthy male New Zealand rabbits were used in the experiment. Middle ear mastoid bone of animal model was completely exposed by surgeon with conventional method, and then Pulse CO 2 laser with a wavelength of 10.6μm and pulse lengths of about 10ms was used to carry out the opening surgery. Laser fluence was 8.3 J/cm 2 with a repetition rates of 60 Hz, the beam diameter was 1.0 mm. After opening surgery, whole middle ear mastoid bone was obtained and processed with traditional histological method, the morphology changes and thermal damage around the opening window were examined by light microscope. Total operation time and light irradiation time were recorded. It showed that pulse CO 2 laser is suitable for the fenestration operation in middle ear bone, and this no-touch technique not only can obtain the similar outcome as traditional methods, but also present a lot of advantages compared to the traditional methods. With the development of laser technology and the appearance of relative instruments, especially when the thermal damage was efficiently controlled, fenestration operation in ear with laser systems will be possible in near future.