The CN groups of polyacrylonitrile (PAN) can strongly adsorb silver ions. The possibility of using this attraction as a layer-by-layer (LBL) self-assembly driving force was investigated. Firstly, the surface of the PAN nanofibrous mats was modified by silver ions to make sure it was positively charged. Then oppositely charged ovalbumin (OVA) and silver ions in aqueous media were alternatively deposited onto the surface of the obtained composite mats by layer-by-layer self-assembly technique. The morphology of the LBL films coating mats was observed by field emission scanning electron microscope (FE-SEM). The deposition of silver ions and OVA was confirmed by X-ray photoelectron spectroscopy (XPS) and wide-angle X-ray diffraction (XRD). The thermal degradation properties were investigated by thermo-gravimetric analysis (TGA). Besides these, the cytotoxicity and antibacterial activity of the prepared mats were studied via flow cytometry (FCM) and inhibition zone test, respectively. The results showed that the composite mats after LBL self-assembly processing exhibited improved thermal stability, slightly decreased cytotoxicity, and excellent antibacterial activity against Escherichia coil and Staphylococcus aureus.