Abstract: The nidamental gland of cuttlefish was used as experimental material, and the single factor experiments and response surface test were used to optimize the extraction process of nidamental gland glycoprotein from cuttlefish. Then the glycoprotein obtained under that was separated and purified. And its purity was analyzed by electrophoresis, its protein and toted sugar content were follow researched. The results showed that the best extraction process of nidamental gland glycoprotein was:extraction temperature 24℃, extraction time 3.6 h, the NaOH concentration 0.4 mol/L, the ratio of material to liquid 1:20 g/mL and the ultrasonic power 200 W. Under the condition of the verification tests, the actual yield of nidamental gland glycoprotein was 16.13%±0.16%. Purified by DEAE-52 celloulus ion exchange column chromatography and Sephacryl S-300HR column chromatography was to obtain pure glycoprotein. The electrophoresis analysis showed that the purity of the purified glycoprotein reached electrophoresis purity, and the protein content was 65.53%±0.8%, and the sugar contents was 31.74%±1.1%. The yield was 0.91% of the nidamental gland to the outer film material.