This paper evaluates the influence of different defocus irradiation conditions on bone hard tissue ablation by pulsed CO2 laser. Bovine shank bones in vitro were used in this experiment which were put on a PC-controlled motorized linear drive stage and moved repeatedly through the focused beam. Work distance was adjusted to obtain a beam spot size of about 510 μm on tissue sample before and after focus plane respectively. The wavelength of pulse CO2 laser was 10.64 μm, pulse repetition rate was 60 Hz, and the energy density is 5～45 J/cm2. The moving speed of the stage was 20 mm/s, scanning times was 6. After irradiation, the incision morphology was observed by naked eye and confocal microscopy. The geometry measurement of the incision was also taken. It was showed that pulsed CO2 laser can be used to cut hard bone tissue, and the defocus irradiation condition has an important influence on ablation effect. In order to obtain a narrow-and-deep incision and high ablation rate, one can locate the beam focus slightly under the bone surface.