A novel physical blend method was developed to accelerate the self-assembly process of silk fibroin (SF) solution into porous and nanofibrous hydrogel by temperature-sensitive copolymer. Silk-based hydrogel was firstly achieved through blending SF solution with copolymer aqueous solution and then removed the copolymer from blend solution by heat treatment (50 degrees C) after 24 h hydrogelation. Copolymer molecules would interact with SF molecules resulting in reduction of copolymer micelles, which further affect the hydrogelation of SF solutions. Copolymers could be separated from blend solution by heat treatment under an acceptable temperature (50 degrees C), especially the copolymer(2). Fourier transform infrared (FTIR) and X-ray diffraction showed the blending of copolymer significantly accelerated the self-assembly of SF into physically crosslinked beta-sheet crystals at room temperature which led to the sol-gel transition. Results from DTA and X-ray diffraction showed that the effect of copolymer on crystalline structure of SF in silk-based hydrogel was very weak. SF molecules transformed from distributed globular nanoparticles to nanofilaments clustered during hydrogelation, resulting in the porous and nanofibrous structure of silk-based hydrogel. Furthermore, silk-based hydrogel was prepared in aqueous solution avoiding organic solvents and harsh processing conditions, suggesting that this silk-based hydrogel could be a potential candidate scaffold for biomedical applications. (c) 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 127: 2019-2024, 2013