In this study, Box–Behnken response surface experimental design of response surface methodology (RSM) was adopted to investigate the simultaneous effects of tomato puree (1–3% w/v), casein (0.4–0.8% w/v) and drying time (10–12 h) on the physical and optical properties of a bio-composite film. Analysis of variance (ANOVA) showed that the experimental data was well-explained by the different models with high coefficient of determination (R2 > 0.90). The second-order polynomial models were fitted to all the response variables and three-dimensional response surface plots and equations were analyzed. It was found that all the process variables significantly affected (P < 0.05) the response variables either linearly or quadratically, whereas the “Lack of Fit” was non-significantly relative to the pure error. On the basis of the ranges of different responses, a total of nine solutions were suggested, out of which, the film with 2.12% w/v tomato puree level, 0.59% w/v casein level and 11.99 h drying time was selected. At this optimum point, thickness, moisture, solubility, moisture absorption rate, water vapor transmission rate, density, transparency, hue angle, chroma value and whiteness index were found to be 0.143 mm, 24.49%, 77.69%, 10.22%, 0.03 g/cm2 t, 1.472 g/mL, 40.72%, 78.70, 11.60 and 64.99, respectively, with a desirability value of 0.61. It can be concluded that the developed films with desirable characteristics can be successfully prepared and effectively applied in the food packaging industry.