The invention relates to an automatic focusing method of an image acquisition device. The method comprises the steps as follows: (a) an automatic focusing area is selected in the picture to be shot, and a focusing weighing is set for each focusing area in the picture; (b) a lens is moved to a relative position, and an image is obtained at the relative position; (c) the evaluation of estimate of the image is calculated according to the selected automatic focusing area and the focusing weighing of each focusing area; (d) the evaluation of estimate and the relative position of the corresponding lens are stored; (e) the steps (b)-(d) are repeated to acquire a plurality of images; the relative position of the lens corresponding to the image with the maximum evaluation of estimate is obtained according to a plurality of evaluations of estimate corresponding to a plurality of images and the relative position of a plurality of lenses, and the shooting position is determined; and (f) the lens is moved to the shooting position. In addition, the invention also provides an image acquisition device adopting the automatic focusing method.