Tomato seed protein isolate (TSPI) (3, 4, and 5 g) was used to form protein-based edible films. The incorporation effect of glycerol (GLY) (35, 42.5, and 50% w/w based on the dry weight of TSPI) and tragacanth gum (20% w/w based on the dry weight of TSPI) on the characteristics of TSPI films was investigated. Response surface methodology (RSM) using a central composite rotatable design was used to predict the effects of the TSPI and GLY concentrations on responses. The experimental data were adequately fitted into a linear model for all of the variables examined. The optimum concentrations of TSPI and GLY were 3.925 g and 35% w/w based on the dry weight of TSPI, respectively. Under this condition, the characteristics of TSPI films resulted in the optimum opacity (14.17), water solubility (48.07%), water vapor permeability (1.355 g mm/kPa h m 2 ), total color differences (52.33), tensile strength (3.5 MPa), elongation at break (6.582%), and L* (63.60). TSPI films exhibited a semicrystalline nature, which was proved by the results of SEM and XRD analysis. Fourier transform infrared spectroscopy (FTIR) showed the presence of strong hydrogen bonding between the protein chains and glycerol. The results showed that TSPI had a good film-forming capacity, creating a homogeneous film with good physiochemical properties. However, this study presented some of the characteristics of TSPI films, aiming to provide new research ideas for the wide application and further study of TSPI edible films.