The aim of this study was to develop three-dimensional silk fibroin-based (SF) scaffolds from Thai yellow cocoon “Nangnoi-Srisaket” of Bombyx mori silkworms. To enhance the biological properties of Thai silk fibroin-based scaffolds, type A gelatin, the denature from of collagen having good biocompactibility, was used to blend with silk fibroin. Three-dimensional scaffolds were prepared by two methods; freeze-drying and salt-leaching. Freeze-dried scaffolds were prepared from blended solutions and crosslinked by dehydrothermal and chemicals. It was found that pure silk fibroin scaffolds possessed highly interconnected porous network. For salt-leached scaffolds, the pore size of silk fibroin scaffold structure represented the size of salt crystals used (600- 710μm). After gelatin conjugating, gelatin was partly formed fibers inside the pores of silk fibroin scaffolds resulting in fiber-like structure with highly interconnection. The results on ATR-FTIR and XRD proved that the structure of freeze-dried silk fibroin scaffolds was random coil while that of salt-leached silk fibroin scaffolds was β-sheet. The compressive modulus of freeze-dried scaffolds having high silk fibroin contents (80-100wt%) was about 350 kPa. Swelling ability of freeze-dried scaffolds decreased as increasing silk fibroin content. For salt-leached scaffolds, gelatin conjugating and hydroxyapatite deposition enhanced the compressive modulus of silk fibroin scaffolds. The results on in vitro culture using bone-marrow derived mesenchymal stem cells (MSCs) on freeze-dried silk fibroin showed that silk fibroin scaffolds were more effective to promote cell proliferation than pure gelatin and blended scaffolds. Mouse osteoblast-like cells (MC3T3-E1) culture showed that gelatin blending and gelatin conjugating could enhance the cell proliferation on silk fibroin scaffolds as noticed from the number of cells and the cell morphology on the surface of scaffolds. This was more distinct in the case of conjugated gelatin/silk fibroin scaffolds. The results implied that Thai silk fibroin looked promising to be applied in tissue engineering.