The aim of this paper is to investigate and optimize the preparation of scutellarin (SCU)-loaded HP-β-CD/chitosan (CS) nanoparticles (CD/CS-SCU-NPs). CD/CS-SCU-NPs were prepared by ionic cross-linking method and the process and formulation variables were optimized using response surface methodology (RSM) with a three-level, three factor Box–Behnken design (BBD). The independent variables were the added amounts of CS, sodium tripolyphosphate (TPP) and Pluronic F-68 during the preparation. Dependent variables (responses) were particle size and entrapment efficiency. Mathematical equations and respond surface plots were used to correlate independent and dependent variables. The preparation process and formulation variables were optimized to achieve minimum particle size and maximum entrapment efficiency by calculating the overall desirability value (OD). The optimized NP formulation was characterized for particle size, PDI, zeta potential, entrapment efficiency and in vitro drug release. According to the results, an optimized CD/CS-SCU-NP formulation was prepared. Results for particle size, PDI, zeta potential and entrapment efficiency were found to be around 200 nm, 0.5, 25 mV, and 70% respectively. For in vitro study, the release of SCU from the NPs exhibited a biphasic release and was in accordance with Higuchi equation. The optimized preparation was simple with the probability for industrialization. The combination use of RSM, BBD and overall desirability values could provide a promising application for incorporating CD into CS nanoparticles as drug delivery carrier and help develop lab-scale procedures.