A new all-aqueous and green process is described to form three-dimensional porous silk fibroin matrices with control of structural and morphological features. Silk-based scaffolds are prepared using lyophilization. Gelatin is added to the aqueous silk fibroin solution to change the silk fibroin conformation and silk fibroin-water interactions through adjusting the hydrophilic interactions in silk fibroin-gelatin-water systems to restrain the formation of separate sheet like structures in the material, resulting in a more homogenous structure. Water annealing is used to generate insolubility in the silk fibroin-gelatin scaffold system, thereby avoiding the use of organic solvents such as methanol to lock in the beta-sheet structure. The adjusting of the concentration of gelatin, as well as the concentration of silk fibroin, leads to control of morphological and functional properties of the scaffolds. The scaffolds were homogeneous in terms of interconnected pores, with pore sizes ranging from 100 to 600 microm, depending on the concentration of silk fibroin used in the process. At the same time, the morphology of the scaffolds changed from lamellar sheets to porous structures based on the increase in gelatin content. Compared with salt-leaching aqueous-derived scaffolds and hexafluoroisopropanol (HFIP)-derived scaffolds, these freeze-dried scaffolds had a lower content of beta-sheet, resulting in more hydrophilic features. Most of gelatin was entrapped in the silk fibroin-gelatin scaffolds, without the burst release in PBS solution. During in vitro cell culture, these silk fibroin-gelatin scaffolds had improved cell-compatibility than salt-leaching silk fibroin scaffolds. This new process provides useful silk fibroin-based scaffold systems for use in tissue engineering. Furthermore, the whole process is green, including all-aqueous, room temperature and pressure, and without the use of toxic chemicals or solvents, offering new ways to load bioactive drugs or growth factors into the process.