Tobacco mosaic virus (TMV) was electrospun with polyvinyl alcohol (PVA) into continuous TMV-PVA composite nanofibers to form a biodegradable nonwoven fibrous mat as an extracellular matrix (ECM) mimetic. Morphological characterizations by electron microscopy showed that the addition of varying amounts of TMV resulted in homogeneous nanofibers without phase separation and did not change the diameter of the composite nanofibers. The orientation of TMV in as-spun fibers could be readily controlled and post-processing of the nonwoven TMV-PVA mat significantly improved its water resistance. In addition, tensile tests were performed on individual nanofibers, which revealed that the TMV-PVA composite nanofibers achieved a comparable Young's modulus as PVA nanofibers. Since the modification of TMV is readily achieved via genetic or chemical methods, this process offers a facile way to incorporate a variety of functionalities into polymer nanofibers. As a demonstration of its potential as ECM mimetic, a mutant TMV containing RGD peptide was co-spun with PVA and the resulting fibrous substrates were used to promote cell growth.