To assess the characteristics of optic nerve impairment between neuromyelitis optica spectrum disorders (NMOSD) and multiple sclerosis (MS) patients by fractional anisotropy (FA). Thirty-one NMOSD patients, 25 MS patients, and 17 heathy controls (HC) who underwent optic nerve DTI were included. The optic nerves of the NMOSD and MS patients were divided into vision-impaired (VI) subgroups and normal-appearing (NA) subgroups according to visual status, respectively. FA values were measured in the anterior, middle, and posterior segments of each intraorbital optic nerve. FA values in VI NMOSD were significantly decreased in the whole optic nerve, especially the posterior segment of the optic nerve (p < 0.001). FA values measured in the anterior and middle segments of the optic nerve in VI MS were significantly decreased as compared to those in the HC (p < 0.05). Between NMOSD and MS, FA values in the posterior segment of the optic nerve showed significant differences (VI NMOSD vs. VI MS, 0.458 ± 0.097 vs. 0.568 ± 0.098, p < 0.001; NA NMOSD vs. NA MS, 0.568 ± 0.098 vs. 0.600 ± 0.085, p = 0.041, respectively). The signal intensity ratio (SIR) in the posterior segment of the optic nerve was significantly increased in VI NMOSD as compared with VI MS (p = 0.002). The combination of SIR and FA for distinguishing VI NMOSD from VI MS resulted in sensitivity, specificity, and positive and negative predictive values of 86.49%, 80.00%, 88.9%, and 76.2%, respectively. FA could quantify the characteristics of NMOSD- and MS-related optic nerve impairment. DTI was a simple and effective imaging tool to differentiate between the two. • NMOSD-related optic nerve impairment is extensive, often greater than half of the optic nerve, with the most significant involvement of the posterior segment of the optic nerve. • MS-related optic nerve impairment is more limited than NMOSD, and anterior and middle optic nerve involvement is common. • Optic nerve DTI is a convenient and effective imaging tool that can help characterize NMOSD and MS.