In this work, the individual and interaction effects of three key operating parameters of the photocatalytic disinfection process were evaluated and optimized using response surface methodology (RSM) for the first time. The chosen operating parameters were: reaction temperature, initial pH of the reaction mixture and TiO2 P-25 photocatalyst loading. Escherichia coli concentration, after 90 minutes irradiation of UV-A light, was selected as the response. Twenty sets of photocatalytic disinfection experiments were conducted by adjusting operating parameters at five levels using the central composite design. Based on the experimental data, a semi-empirical expression was established and applied to predict the response. Analysis of variance revealed a strong correlation between predicted and experimental values of the response. The optimum values of the reaction temperature, initial pH of the reaction mixture and photocatalyst loading were found to be 40.3 °C, 5.9 g/L, and 1.0 g/L, respectively. Under the optimized conditions, E. coli concentration was observed to reduce from 107 to about 11 CFU/mL during the photocatalytic process. Moreover, all these results showed the great significance of the RSM in developing high performance processes for photocatalytic water disinfection.