Despite the abundant sunlight, vitamin D deficiency is prevalent in South Asian countries including Bangladesh. Information on vitamin D level is insufficient in adults particularly in female garment workers in Bangladesh. This study was designed to evaluate the status of vitamin D, parathormone (PTH), calcium, and alkaline phosphatase (ALP) among the female garment workers in Bangladesh. Blood samples were collected from female garment workers (n = 40, case group) and general female workers (n = 40, control group) in Dhaka. Serum vitamin D, PTH, calcium, and ALP were measured by chemiluminescence microparticle immunoassay. The mean level of vitamin D was significantly (p < 0.001) lower in case (14.2 ± 2.6 ng/mL) than in the control (22.4 ± 2.4 ng/mL) group. No significant difference was found at mean of PTH and calcium between case (33.9 ± 17.2 pg/mL; 9.1 ± 0.6 mg/dL, resp.) and control (35.9 ± 16.3 pg/mL; 9.3 ± 0.6 mg/dL, resp.) group. The mean ALP in case (117.2 ± 14.4 U/L) group was significantly (p < 0.001) higher than the control group (80.5 ± 30.6 U/L). Overall, PTH level did not show significant correlation with vitamin D. However, calcium and ALP levels showed a significant positive (p < 0.05) and negative (p < 0.001) correlation with vitamin D, respectively. This study indicates a high prevalence of vitamin D deficiency in the female garment workers in Bangladesh.