In this study, wheat gluten fibers loaded by the azathioprine were fabricated and their physicochemical properties were studied. The nanofibers were characterized by using the scanning electron microscopy (SEM), Fourier transformed-infrared spectroscopy, X-ray diffractometry, thermogravimetric, and differential thermal analysis. The SEM images showed that the addition of polyvinyl alcohol decreased the fibers' diameters. The diameters of 77.14 ± 14.11 nm were obtained for the wheat gluten–polyvinyl alcohol loaded drug fibers. In addition, the cell viability evaluation conducted by the MTT assay proved no cytotoxicity of the prepared nonofibers at concentrations up to 4 mg/mL to human umbilical vein endothelial cell.