Thiamine (vitamin B1) was the first B vitamin which has been identified. It serves as a cofactor for several enzymes involved in energy metabolism. The laboratory test against thiamine deficiency can be done by measuring thiamine levels in the blood. The aim of this study was to identify the stability and the binding activity characters of TBP. The equilibrium dialysis technique was used to see the factors affecting the bond between TBP and thiamine. The MBTBP concentration of post-chromatographic affinity resulted from dilution of lyophilisate was stable for 30 days at -20°C and 3 days at 4°C. The optimal pH for binding MBTBP to thiamine was 7.5. Alkylation with iodoacetic acid decreased the binding capacity of TBP which suggested the presence of a–SH or imidazol group in its active site. The importance of disulfide bridge was proven by decreasing of Thiamine binding capacity of TBP after β-mercaptoethanol treatment. This binding activity was also affected by oxidizing agents, but it was less affected by calcium ions and heavy metals. © 2020 Phcogj.Com.