In spite of the numerous advantages of natural polymer nanofibers as biomedical materials, they are suffering due to their poor water resistance and tensile strength which largely restrict their applications. Here, we present a novel assembled protein nanofabric prepared by incorporating compact zein nanoparticles into electrospun hordein networks. The effects of zein content on the structure, morphology and properties of assembled protein fibers were investigated in detail. The results revealed that zein particles were well dispersed in hordein networks while maintaining their compact structures, which in one aspect acted as the plasticizer to decrease the strong hydrogen bonding interactions among extended hordein molecules, leading to the accelerated continuous electrospinning and narrowed diameter distribution of composite fibers. In another, hydrophobic zein nanoparticles also acted as the reinforcing filler in the flexible hordein matrix. The fibers with a zein content of 30 wt% exhibited the stable assembled network structure and significantly improved tensile strength and wet stability in both water and ethanol. The release experiment indicated that such fibers with a 3D porous structure could serve as the carrier for controlled release of incorporated bioactive compounds into phosphate-buffered saline (PBS). In addition, they were stable in simulated gastric fluid and pepsin resistant, whereas they could be digested in a simulated intestinal environment to gradually release the incorporated compounds where they are normally absorbed. These fibers also demonstrated low toxicity in both human colon carcinoma (Caco-2) and primary epidermal keratinocyte (NHEK) cell cultures. Therefore, the novel assembled fabrics showed promise as a three-dimensional delivery vehicle of bioactive compounds for wound healing and food industry applications.