Objective: Subclinical vitamin B12 deficiency and adverse health outcomes are of general concern. Current biomarkers of vitamin B12 status are not always satisfactory to decide on a deficiency state. Recently, holotranscobalamin (holoTC) has been proposed as a useful alternative indicator of vitamin B12 status, however studies on its value in diagnosing cobalamin deficiency have not come to a conclusion yet. The purpose of this study is to investigate the usefulness of holoTC measurement together with total vitamin B12 measurement in diagnosing cobalamin deficiency, in a cross-sectional analysis. Material and Method: Four hundred volunteers were grouped according to vitamin B12 levels as vitamin B12 deficient (vit B12<193 pg/ml, n=168), borderline (vit B12<193-300 pg/ml, n=100) and controls (vit B12>300 pg/ml, n=132). These groups were divided into two subgroups (A and B) according to holoTC cut-off value (35 pmol/L). The diagnostic efficacy of vitamin B12, holoTC and a combination of both measures were evaluated. Serum folate and homocysteine (Hcy) were used as indicators of vitamin B12 deficiency. Results: Significantly higher Hcy and lower folate levels were observed in both vitamin B12 deficient and borderline B12 groups provided that holoTC levels were low. Conclusion: Evaluation of vitamin B12 measures together with holoTC measures provides a more accurate diagnosis, especially in patients with borderline-B12 concentrations.