Background: It has been suggested that hypovitaminosis D is associated with the development of preeclampsia. This study aimed to study the relationship between preeclampsia and various vitamin D serum biomarkers including 25-hydroxyl vitamin D [25(OH)D], vitamin D-binding protein (VDBP), and bioavailable and free 25(OH)D. Methods: This prospective study was conducted with 17 patients with preeclampsia and 38 normal pregnant women as the control group. Total serum 25(OH)D and VDBP concentrations were measured. The levels of bioavailable 25(OH)D and free 25(OH)D were also calculated. Two single nucleotide polymorphisms (rs4588 and rs7041) of the GC gene encoding VDBP were analyzed. Results: VDBP was significantly increased in the preeclampsia group compared to the normal pregnancy group (454.2 vs. 403.4 ng/mL; p = 0.036). When the preeclampsia patients were analyzed by dividing them into early-onset and late-onset, there was no significant difference in the serum vitamin D biomarkers levels. Also, when preeclampsia patients were classified into three subgroups of 2 days, 2–7 days, and >7 days from diagnosis to delivery, free 25(OH)D concentrations were significantly increased in the 2–7 days subgroup compared to the other subgroups (3.5 vs. 6.6 vs. 3.1 pg/mL; p = 0.032). The GC genotype and allele frequency showed no statistically significant different distribution between the preeclampsia and normal pregnancy groups. Conclusions: In the present study, the serum VDBP levels were significantly higher in the patients with preeclampsia than in the normal pregnancy group. Thus, among various serum vitamin D biomarkers, increased VDBP could be associated with the onset and pathogenesis of preeclampsia.