Based on the previous ultrafiltration separation of chitosan hydrolysate, the technical conditions were investigated, for the nanofiltration separation of ultrafiltrate using roll-type nanofiltration membrane with a relative molecular weight cutoff of 500 Da. The effects of process parameters including the operating pressure and temperature, as well as the pH value of filtrate on the separation and purification of chitosan hydrolysate were studied by indicators of membrane flux, glucosamine removal rate, conductivity and pH value. The optimal operating parameters for nanofiltration were as followed: operation presure 0.50 MPa, operation temperature 35 ℃, and pH 3.0. The final product with 1.21% glucosamine content, 0.15% ash content and 70.25% chitooligosaccharide of 3～6 glucosidic bonds was obtained, and the product yield was 70.12%.