Background: Vitamin D deficiency is associated with a range of systemic diseases including ocular disorders. The objective of this study is to measure tear vitamin D levels and investigate the correlation between serum and tear vitamin D levels. Methods: A total of 48 healthy volunteers without any systemic and ocular disease were recruited for this observational cohort study. Serum was collected using clot activator coated Vacutainer® Plus tubes. Tear fluid was collected using Schirmer's strips. Serum and tear total 25-hydroxyvitamin D levels were measured by competitive chemiluminescent ELISA and the correlation between the levels were studied. Results: The measured serum 25-hydroxyvitamin D level ranged between 3.3 and 27.5 ng/ml (Mean ± SEM, 9.4 ± 0. 7 ng/ml; Median 8.4 ng/ml). Significantly (p < 0.0001) higher level of 25-hydroxyvitamin D was detected in the tears (Mean ± SEM, 17.0 ± 1.6 ng/ml; Range 3.2-45.8 ng/ml; Median, 16.3 ng/ml) compared to serum. An average of~2 fold (Mean ± SEM, 1.9 ± 0.2; Range 0.4-5.8; Median, 1.7) higher 25-hydroxyvitamin D was observed in tears compared to serum in the subjects. In addition, a positive correlation was observed between serum and tear 25-hydroxyvitamin D levels (r = 0.5595; p < 0.0001). Conclusions: A higher level of 25-hydroxyvitamin D was observed in the tear fluid compared to that of the serum. It would be beneficial to consider tear vitamin D levels to study its role with reference to ocular surface diseases.