Defocusing phenomenon induced by the change of the work distance in the irradiating process of biological tissue has an important influence on the effect of hard tissue ablation with pulsed lasers. Fresh bovine shank bone in vitro used in the experiment is put on a computer-controlled motorized linear drive stage and is moved repeatedly with the speed of 5.5 mm/s. A parallel beam of pulsed CO2 laser passes through a mobile focus lens and irradiates the sample surface vertically. Through changing the position of focusing lens, the focus position is altered and dynamic focusing is realized. The laser wavelength is 10.64 μm, the power is 6 W, the pulse repetition rate is 420 Hz, the spot size is about 200 μm, and the water spraying with a speed of 5.5 mL/min is used. The surface morphology and microstructure of ablation grooves are examined by scanning electron microscope (SEM). The crater depth is measured with optical coherence tomography (OCT). The results show that compared with defocusing condition, dynamic focusing condition can enhance the ablation rate and produce more irregular surface morphology and microstructure. The ablation depth increases with the increase of equivalent pulse number and the ablation rate decreases.