A novel Starch/Poly (vinyl alcohol)/nano-Titanium dioxide (ST/PVA/nano-TiO2) biodegradable nanocomposite film was prepared by homogeneously dispersed TiO2 nanoparticles in different ratios of starch/PVA-based materials, via a solution casting method. Glycerol was used as plasticizer. The mechanical and thermal properties of films were studied using tensile and perforation strength tests and thermogravimetric analysis (TGA). In order to determine the effective parameters on mechanical properties of prepared films, a general full factorial experimental design approach was used. ST/PVA/TiO2 nanocomposites were also characterized by scanning electron microscopy (SEM). The results of mechanical analysis showed that ST/PVA films with higher content of PVA had much better mechanical properties. In thermal analysis it was found that addition of TiO2 nanoparticles have considerable effects on the thermal stability of the films. This enhancement of thermal stability can be attributed to an improvement in the interfacial adhesion and compatibility between the nano- TiO2 and matrix, due to the treating effect of plasticizer and film forming process. SEM micrographs, taken from the fracture surface of samples, illustrated a quite uniformly dispersion of TiO2 nanoparticles in the matrix. Moreover, addition of PVA to the composition of film made it softer and more flexible.