Diabetic autonomic neuropathy (DAN) is one of the most disabling complications of diabetes mellitus. This study aimed to evaluate a special pupillary analysis for the early diagnosis of DAN by investigating pupil responsiveness under different conditions. Images of the pupils of 40 type 2 diabetic patients, who were divided into two (subclinical and nonproliferative diabetic retinopathy [NPDR]) groups using the results of fundus fluorescein angiography, and 20 healthy subjects were taken under mesopic, photopic, and pharmacologically dilated conditions. The pupil diameters, constriction ratio (CR), and dilation ratio (DR) were computed and calculated from the images. The mean mesopic pupil diameters (D1) of the control group, the subclinical group, and the NPDR group were 6.02 ± 0.48, 5.87 ± 0.99, and 4.95 ± 1.12 mm, respectively. The mean photopic pupil diameters (D2) were 3.40 ± 0.33, 3.37 ± 0.31, and 3.25 ± 0.47 mm, respectively. The mean pharmacologically dilated pupil diameters (D3) were 7.37 ± 0.59, 6.91 ± 1.00, and 5.49 ± 1.24 mm, respectively. The mean CR of the three groups were 0.43 ± 0.07, 0.41 ± 0.10, and 0.32 ± 0.14, respectively, and the mean DR were 0.23 ± 0.08, 0.19 ± 0.13, and 0.11 ± 0.05, respectively. There were statistically significant differences between the three groups with regard to the data of dark-adapted pupil diameter, dilated pupil diameter, CR, and DR ( p < 0.05). Autonomic pupillary dysfunction occurs early in type 2 diabetic patients. Abnormal pupil responses might be due to a dysfunction of pupillary nerve fibers. In addition, myogenic abnormalities are a causative factor to produce small D1 and DR. Pupillary diameters under mesopic and pharmacologically dilated conditions and the CR and DR of the pupillary function can be reliable parameters to evaluate autonomic neuropathy in type 2 diabetes mellitus patients.