The objective of this study was to optimize the particle size and encapsulation efficiency of chitosan nanoparticles loaded with 5-Fluorouracil (5-FU) by response surface methodology. Nanoparticles were prepared by ionic gelation method from chitosan and penta sodium triphosphate (TPP) at different combinations of chitosan viscosity, chitosan concentration and chitosan/TPP mass ratio according to the Box-Behnken experimental design. The particle size and encapsulation efficiency of prepared particles were measured by dynamic light scattering and UV spectroscopy, respectively, and the obtained data were subjected to multiple linear regression analysis followed by multi-attribute utility analysis to obtain a model for prediction of the optimum response. The optimum conditions for the production of 5-FU loaded chitosan nanoparticles were found to be low viscosity chitosan 0.5-1 mg/mL, middle viscosity chitosan 0.5-0.8 mg/mL and high viscosity chitosan 0.5-0.75 mg/mL and chitosan/TPP mass ratio of 4 or 6, yielding nanoparticles at the average diameter range of 114-188 nm and encapsulation efficiencies between 42-55%.