An autofocusing method, based on the characteristic model of the number of pixels changing with the defocus distance, was proposed to quickly obtain a clear fluorescence image of a micropore digital polymerase chain reaction (PCR) chip and overcome the issue of time-consuming calculation in the traditional method for autofocusing of the microscale array unit. Thereafter, fluorescent images of three microarray digital PCR chips equidistant from each other were selected. The threshold was calculated through an adaptive window. The pixels in the window area with values larger than the threshold were counted. The defocus distance was calculated by substitution in the characteristic function. The defocus direction was determined by the pixel values of the three positions and then focusing was carried out. This method uses only statistics of values larger than the threshold of the 13×13 pixels in the focusing window and completes focusing in only four steps. The focusing results satisfied the requirements of subsequent calculations. Compared with the traditional mountain climbing method, the number of focusing steps was decreased by 51.89% on average upon the implementation of the microarray-type digital PCR chip accurate focusing. The proposed autofocus method overcomes the shortcomings of contemporary algorithms, such as extensive computation and tedious focusing steps, and provides an accurate and rapid focusing method. It minimizes the exposure time of the sample, thereby reducing fluorescence quenching, and provides more original and accurate images for subsequent calculations.