BACKGROUND To determine the association between serum 25(OH)D and dry eye syndrome (DES) incidence. This study was also designed to determine whether serum 25(OH)D levels were associated with ocular parameter of DES patients. MATERIAL AND METHODS This is a case-control study with 70 DES cases and 70 healthy controls. Clinical data included body mass index (BMI, kg/m²), smoking history, diabetes, and blood pressure. Serum 25(OH)D was chosen as the main parameter and reflected the level of vitamin D. The DES parameters included ocular surface disease index (OSDI) scales, tear film breakup time (TBUT) and Schirmer test I. The differences in each parameter between case and control groups were detected and the association of serum 25(OH)D and DES parameter were detected. RESULTS It was shown that 25(OH)D levels were lower in patients with DES than in healthy controls. When the 25(OH)D levels was stratified, vitamin D deficiency was more common in the DES cases. In advanced studies, it was found that there were statistically significant associations between serum 25(OH) D levels and the Schimer test, TBUT, and OSDI scales. CONCLUSIONS A significant association between serum 25(OH)D level and DES incidence was detected in this study. Considering the relatively small sample size of this study, larger studies are needed in the future.