The autofocus is a key technique for automatic reconstruction in digital holography, and the performance of the focusing criterion function influences the effect of the autofocus greatly. After analyzing and researching existing focusing criterion functions and studying the theories about image definition, for the digital holograms of marine microorganism, a new focusing criterion function is proposed. The method proposed is based on features that local standard deviation distribution is able to reflect image detail information and standard deviation which is taken as measurement of discrete degree has high robustness. It makes the discrete degree of reconstruction image detail as image definition index, and makes reconstruction distance corresponding to maximum of the definition as the best distance to accomplish the autofocus. Simulation results illustrate that compared with the existing methods, the proposed method has the stronger single peak, higher sensibility and signal to noise ratio (SNR) and better unbiasedness. In addition, it has strong universal and practical for the digital holograms of marine microorganism. So it can satisfy the requirements of automatic reconstruction of the marine microorganism holograms.