This paper proposes a new autofocusing method for microbiological specimens, such as cells. The proposed focusing method used a quick focus estimation named "depth from diffraction", based on a diffraction pattern in a defocused image of a biological specimen. Since this method can estimate the focal position of the specimen from only a single defocused image, it can easily realize high-speed autofocusing. To demonstrate the proposed method, continuous focusing was applied to focus tracking of a swimming paramecium, in combination with two-dimensional tracking. This allowed three-dimensional tracking of the paramecium for 75 s to be successfully demonstrated.