Background: Despite the potential role of vitamin D in the acute phase of COVID-19, studies on its prevalence and association with long COVID severity are limited, leaving the relationship between vitamin D levels and long COVID symptoms unclear. Aim: The objective of this study was to investigate the prevalence of vitamin D deficiency in patients with fatigue or neuropsychiatric symptoms of long COVID and its correlation with the severity of symptoms. Methods: A cross-sectional, single-center study was conducted at Thammasat University Hospital, Thailand. The study recruited participants between the ages of 18 and 60 years who had received a diagnosis of COVID-19 and had at least one long COVID symptom, including fatigue or neuropsychiatric symptoms. Vitamin D level was collected, and clinical severity was assessed using the Chalder Fatigue Scale [CFQ-11], Depression, Anxiety and Stress Scale-21 Items [DASS-21], Pittsburgh Sleep Quality Index [PSQI], Addenbrooke's Cognitive Examination III [ACE], and Trail Making Test A & B [TMT-A and TMT-B]. Results: A total of 82 patients were recruited; most were female [78%], with a mean age of 34.2 years. Most participants had a vitamin D deficiency [<20 ng/mL, 73.2%], accounting for 23.2% vitamin D insufficiency [20-30 ng/mL], and 3.6% had an adequate vitamin D level. Only gender was associated with vitamin D levels. Multivariable analysis demonstrated that the vitamin D level was not correlated with all clinical outcomes, including total CFQ, total DASS, DASS depression, DASS anxiety, total PSQI, total ACE score, and total TMT scores. Conclusion: The prevalence of vitamin D insufficiency and deficiency was high in patients with long COVID, with female gender serving as predictors of lower vitamin D levels. However, no associations were found between vitamin D level and fatigue syndrome, depression, anxiety, sleep problems, or cognitive function.