Silk fibroin nanofiber hydrogels mimicking the extracellular matrix structure have gained increased attention in the field of tissue engineering. In this study, an amorphous silk fibroin nanofiber solution was prepared using a new dissolving technique. The keys to the dissolving method include the preparation of nanofibrous silk film by dissolving silk in a CaCl2-Formic Acid (Ca-FA) solution with subsequent dissolution in a CaCl2-Ethanol-H2O ternary solution at room temperature. The resulting solution was primarily composed of native silk nano/microfibers, which primarily form amorphous structures. The amorphous silk fibroin nanofiber solution was further processed into an injectable hydrogel through a simple physical blend with polyethylene glycol (PEG). The nanofiber structure and PEG accelerated silk gelation, and the fastest gelation time was 150 s at concentrations of 5% silk and 85% PEG. The morphology, secondary structure, and rheological properties of injectable silk fibroin nanofiber hydrogels were subsequently characterized. Silk gelation was accompanied by a structural transition from random coil to β-sheet, and the resulting injectable hydrogel had an irregular lamellar structure. The antibiotic vancomycin (VANCO) was loaded in the injectable hydrogel, and the release of VANCO from the injectable hydrogel was assessed at pH values of 4.5 and 7.4. The VANCO-loaded injectable silk fibroin nanofiber hydrogel exhibited superior antibacterial properties against bacteria. A cytotoxicity test preliminarily demonstrated that the VANCO-loaded injectable silk fibroin nanofiber hydrogel had good biocompatibility for applications in tissue engineering.