In this study, regenerated silk fibroin (RSF)/tetramethoxysilane (TMOS) composite electrospun fibers with improved hydrophilicity were successfully prepared by electrospinning method, which was superior for fibroblast attachment. The electrospinning process caused adjacent fibers to weld at contact points, as confirmed by scanning electron microscope (SEM) analysis. Fourier transform infrared spectroscopy (FTIR) showed that TMOS has been well incorporated into the silk fibroin electrospun fibers. Thermogravimetric analysis (TGA) was carried out to quantify the water content in RSF/TMOS composite electrospun fibers. The cytotoxicity and L929 adhesion of three-dimensional RSF/TMOS fibrous biocompoaites were investigated and compared to pure RSF membrane. The water contact angle of RSF/TMOS nanofibrous composites showed a sharp decrease compared to the pure RSF electrospun fibers, which has a great effect on the early stage of cell attachment behavior due to an relatively enhanced hydrophilicity.