A new auto-focusing algorithm based on Depth from Focus (DFF) was presented according to the properties of the automatic iris imaging system. The algorithm used the ULFM value of the image as the sharpness evaluation function on the basis of the fact that the automatic iris cameras have small depth of field, and employed a two-step auto-focusing strategy by combining coarse focusing with fine focusing. Experiments of automatic iris imaging system prove that the auto-focusing algorithm proposed is of high precision and strong robustness. The searching time meets the needs of real-time auto-focusing. The system can accurately acquire clear iris images for recognition with a work scope of 0.5 meter to 2 meters and the rate of successful focusing is higher than 95%.