A series of poly(vinyl alcohol) (PVA)/regenerated silk fibroin (RSF)/nano-silicon dioxide (nano-SiO2) blend films were prepared by solution casting method, in which nano-SiO2 was obtained via sol–gel process. The structure, properties, and morphology of the films related to the compatibility were investigated by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). XRD peaks of PVA/RSF/nano-SiO2 (1.0 wt %) blends decreased in intensity indicated that formation of PVA and RSF crystal lattices was hindered by nano-SiO2 particles. FTIR spectroscopy analysis of PVA/RSF/nano-SiO2 films confirmed that both Si–O–C linkage and hydrogen bonding were formed among PVA, RSF, and nano-SiO2. SEM showed that there was no obvious phase separation in PVA/RSF/nano-SiO2 (1.0 wt %) film although small uniform blur particles can still be found. In addition, TEM showed nano-particles were well dispersed through the PVA/RSF polymer matrix. Besides, the observed shift in glass transition temperatures (Tg) and improvement in thermal properties of composite films suggested the enhanced compatibility due to interfacial bonding and intermolecular interactions. Therefore, these results indicated that the compatibility of PVA/RSF was improved effectively by the addition of nano-SiO2.