Abstract: Orthogonal design was used to optimize the binding capacity of Sodium Cholate (SC) and Sodium Deoxycholate (SD) by nine partially depolymerized chitosans obtained from a native chitosan using sodium nitrite (NaNO2). The effects of sodium treatment on depolymerization of chitosan were investigated by measuring the molecular weight, viscosity and reducing sugar of chitosan. Depolymerization for 3 h reduced the molecular weight of the chitosan by 91% (26 kDa) compared to the native chitosan (458 kDa) with a decrease in viscosity of the chitosan solution and increased reducing sugar. The binding capacity of total cholesterol, SC and SD by chitosan samples were measured in vitro by enzymatic and HPLC methods, respectively. The optimized conditions were sodium nitrite (0.4%, w/v), acetic acid (1 and 2%, v/v), chitosan (3%, w/v) and reaction time (1 h) at room temperature, sodium chelate 69 μmol g-1 chitosan and deoxycholate125 μmol g-1 chitosan. The strongest binding capacity of sodium deoxycholate and total cholesterol was observed with partially depolymerized chitosan (average molecular weight 52 kDa) compared to the native chitosan.