In this paper, by the method of threshold segmentation, the characteristic parameters of dot microscopic image were accurately acquired which were used in combination with the instrument measured value to comprehensively evaluate the quality of printing. This experiment presented a threshold segmentation algorithm based on Gaussian function model to fit the gray histogram data of dot microscopic image to find the optimal threshold of the dot image and use this threshold to do binarization processing of image and getting accurate dot parameters. The obtained printing dot area rate by using this method was closer to the measurements throughout the tone range, the segmentation effect was better than the traditional threshold segmentation algorithm. In conclusion, Gaussian fitting threshold segmentation algorithm proposed in this paper is more beneficial to extract the microscopic parameters of the dot image, with high extraction precision and good stability, which provides a theoretical and practical reference for obtaining accurate dot image microscopic parameters.