Fibrous scaffold could provide extracellular matrix (ECM) like structure and desired network for cell growth; however, the mechanical performance of this type uni-structured fibrous scaffold cannot meet the requirement of tissue formation. Therefore, new strategies are needed for form mechanical strength enhancement. In this study, we developed three dimensional double-network structured fibrous scaffold (3D DN-Fs) using self-assembly technology combined with electrospinning technology. Our 3D DN-Fs consists of two types of skeletons: the finer silk nanofibers which can mimic biocompatible ECM structure; and the larger skeletal fibrous layers can greatly improve the mechanical strength and cellular loading ability, and provide good nutrition and excreta delivery system for cell growth. Therefore, our 3D DN-Fs displayed excellent mechanical performance (more than 50% increment), biocompatibility, biodegradability, and a desirable microenvironment for cell growth. More importantly, cultured cells exhibited excellent viability and 3D growth. Our novel strategy greatly enhances the potential application of fibrous scaffold in the biomedical area, such as 3D cell culture and tissue engineering.