Objective To measure serum total vitamin D or 25-hydroxyvitamin D [25(OH)D] levels and status in immune-based optic neuritis (ON) including neuromyelitis optica spectrum disorder (NMOSD)-ON, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD)-ON, autoimmune-ON, and idiopathic-ON and compare them with age- and sex-matched healthy controls. The secondary objective was to analyze the association between serum 25(OH)D levels and ON attack severity (nadir best-corrected visual acuity; nadir BCVA). Materials and Methods This was a single-center, case–control study. We enrolled 59 subjects (19 NMOSD-ON, 6 MOGAD-ON, 11 autoimmune-ON, 23 idiopathic-ON) diagnosed with acute immune-based ON (any ON attacks) over 11 years. Electronic medical records were reviewed and demographic data (age at sampling, sex, aquaporin-4 immunoglobulin (AQP4-IgG); myelin oligodendrocyte glycoprotein immunoglobulin G (MOG-IgG); other biomarkers of autoimmune disorders), ON attack severity (nadir BCVA), and serum 25(OH)D levels in the acute phase of ON were collected. Serum 25(OH)D levels of 236 age- and sex-matched healthy controls were assessed. Results Mean serum 25(OH)D levels were significantly lower in each group of immune-based ON compared with healthy controls ( p < 0.001 for each ON group). However, mean serum 25(OH)D levels were not significantly different between four ON groups (NMOSD-ON, 20.18±5.90 ng/mL; MOGAD-ON, 23.07±4.94 ng/mL; autoimmune-ON, 21.14±5.29 ng/mL; idiopathic-ON, 19.56 ±5.12 ng/mL; p = 0.525). All immune-based ON subjects had vitamin D insufficiency or vitamin D deficiency. The prevalences of vitamin D insufficiency and vitamin D deficiency were significantly higher than in healthy controls in each ON group (both p < 0.05 in each ON group). No associations were observed between serum 25(OH)D levels and ON attack severity (nadir BCVA). Conclusions Thai immune-based ON subjects had lower serum 25(OH)D levels and higher prevalence of vitamin D insufficiency and vitamin D deficiency compared with age- and sex-matched healthy controls. Serum 25(OH)D levels were not associated with ON attack severity (nadir BCVA). We highly recommend that serum 25(OH)D levels be screened in all subjects with acute immune-based ON.