The aim of the study was to examine whether holotranscobalamin is a more sensitive indicator of vitamin B12 deficiency in relation to cobalamin, and whether there is a significant correlation between cobalamin, holotranscobalamin and homocysteine levels. Patients and methods The study included 54 subjects who were divided into two groups according to the findings of homocysteine. The first group included 31 patients with elevated homocysteine values and with neurological symptoms, whereas 23 patients with normal homocysteine values and different diagnoses which represented controls. The cocentrations of cobalamin, holotranscobalamin and homocysteine were measured in serum samples with chemiluminescent microparticle immunoassay (CMIA) (Abott Laboratories) on the Architect i1000SR analyzer ( Abbott Diagnostics , Lake Forest , United States ). The results were processed using MedCalc Software (MedCalc Software, Mariakerke, Belgium). ROC curve was used to test sensitivity and specificity, while Mann-Whitney test and Fisher exact test were used for groups comparision. For testing the connection, correlation coefficient was used. Results The test showed that holotranscobalamin is not a more sensitive indicator of vitamin B 12 deficiency than cobalamin (AUC= 0.511 vs. AUC= 0.596), and that there is a weak connection between holotranscobalamin and cobalamin (r= 0.333). However, between holotranscobalamin, and cobalamin and homocysteine there is no correlation (r= 0.032 vs. r= -0.103). Conclusion Research shows that holotranscobalamin is not a more sensitive indicator of vitamin B 12 deficiency than cobalamin. It can be said that holotranscobalamin and cobalamin, separately or in combination have almost equal diagnostic efficiency in the diagnosis of vitamin B12 deficiency as early indicators.