In this study, negatively charged phosvitin (PV) and positively charged chitosan (CS) were alternately deposited on negatively charged cellulose mats via layer-by-layer (LBL) self-assembly technique. Morphologies of the LBL films coating mats were observed by scanning electron microscope (SEM). Afterwards, in vitro biomimetic mineralization was carried out through incubation of the fibrous mats in a simulated body fluid (SBF) solution for different time. Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) were used to characterize the morphology and structure of the deposited mineral phase on the scaffolds. In addition, the cell culture experiment demonstrated that the scaffolds with the LBL structured films were of good cell compatibility for MC3T3-E1 cells. Moreover, the cell proliferation was affected by the number of deposition layers and the composition of outer-most layer. Confocal laser scanning microscopy (CLSM) and SEM imaging revealed a good performance of cell adhesion and spreading of MC3T3-E1 cells on the surface of biocomposite scaffold. So CS/PV nanofibrous mats were satisfactory for the composite to be used in bioapplications.