Background: In tissue engineering, scaffold characteristics play an important role in the biological interactions between cells and the scaffold. Cell adhesion, proliferation, and activation depend on material properties used for the fabrication of scaffolds.Objective: In the present investigation, we used collagen with proper characteristics including mechanically stability, biodegradability and low antigenicity. Optimization of the scaffold was done by immobilization of ALKALINE PHOSPHATASE on the collagen surface via cross-linking method, because this enzyme is one of the most important markers of osteoblast, which increases inorganic phosphate concentration and promote mineralization of bone formation.Methods: ALKALINE PHOSPHATASE was immobilized on a collagen surface by 1-ethyl-3- (dimethylaminopropyl) carbodiimide hydrochloride, as a reagent. Then, rat mesenchymal stem cells were cultured in osteogenic medium in control and treated groups. The osteogenesis-related genes were compared between treatments (differentiated cells with immobilized ALKALINE PHOSPHATASE/collagen scaffold) and control groups (differentiated cells on collagen surface without ALKALINE PHOSPHATASE) on days 3 and 7 by quantitative real-time PCR (QIAGEN software).Results: Several genes, including ALKALINE PHOSPHATASE, collagen type I and osteocalcine associated with calcium binding and mineralization, showed upregulation in expression during the first 3 days, whereas tumor necrosis factor-α, acting as an inhibitor of differentiation, was down-regulated during osteogenesis.Conclusion: Collagen scaffold with immobilized ALKALINE PHOSPHATASE can be utilized as a good candidate for enhancing the differentiation of osteoblasts from mesenchymal stem cells.