Engineered scaffolds simultaneously exhibiting multiple cues are highly desirable for neural tissue regeneration. Silk fibroin is a promising natural protein material for nerve repair. However, the lack of specific bioactive cues significantly hinders its application. In this study, the electrospun silk fibroin nanofibers with both biochemical and topographical cues were prepared. The alignment of electrospun nanofibers was optimized by controlling the surface linear velocity of a rotating drum. The silk fibroin nanofibers were further functionalized with laminin through covalent binding, confirmed by immunostaining observation. Cell proliferation and neurite outgrowth assays confirmed that the functionalized aligned nanofibers significantly enhanced directional axonal extensions, providing physical and bioactive cues for neurite outgrowth. Furthermore, the tubular scaffolds with longitudinally aligned microchannels were designed by rolling the functionalized silk fibroin nanofibers. The neurite extension across the lumen of the conduit along the direction of the aligned fibers is apparent. These results highlight the ability of laminin-immobilized silk fibroin nanofibers to enhance neurite outgrowth and to control directional neurite extension, providing a useful approach to construct a regenerative microenvironment for nerve repair materials.