Nanoparticles of ultra low molecular weight chitosan (ULMWCS)/hyaluronic acid (HA) were prepared by ion gelation. Three independent variables, namely, ratio of concentration of ULMWCS to HA (CS/HA), pH of solution and stirring time were studied to identify their effects on size, polydispersity and zeta potential of prepared nanoparticles using a Box Behnken design. Results showed that pH and CS/HA have a direct effect on size, while increase of stirring time decreases the size of nanoparticles. Additionally, it was shown that all the independent parameters have direct effects on zeta potential. Also, the minimum polydispersity index was observed at lowest values of CS/HA. The model also predicted that the optimum values are 4.15, 4.14 and 180 (min) for the CS/HA, solution pH and stirring time, respectively. The obtained preparation had a size of 200nm, polydispersity index of 0.37, and zeta potential of 13.0mV.