Abstract: In order to determine the influence of femtosecond laser machining center on ablation depth with micro-scale structure, the effective ablation focal length of laser beam corresponding to the given power was researched, a method for getting the ablated spot array image at laser focus, extracting the image feature in defocus stage and obtaining the effective ablation focus range of laser beam by analyzing the relationship between the image features and the defocus distance was proposed. Firstly, the spot array was ablated on the surface of silicon wafer near the laser beam focus. Secondly, the focal length was gradually reduced downward to capture the spot image of silicon wafer, the average pixel area of spot image and the gray difference of R component between spot target and background were extracted, and the change curves of spot pixel area and gray difference with focal length of laser beam were obtained. Again, the focal length was gradually increased upward, the change curve of spot pixel area and gray difference with focal length of laser beam was extracted and obtained similarly. Finally, combined with the downward defocus threshold (633 μm) and upward defocus threshold (993 μm) of laser beam, under the condition of 20 mW output power, the effective ablation depth of femtosecond laser on silicon wafer surface was determined to be 360 μm. By using median value method, the focal length on the surface of silicon wafer was determined to be 0.823 mm while defocused. Experimental results show that the relationship among spot pixel area, gray difference and laser beam focal length can objectively reflect the variation range of effective ablation focal length of laser beam.