Abstract: In order to provide scientific basis for the comprehensive utilization of high nitrogen waste water from tuna processing industry. Besides, it can provide theoretical guidance for pollution reduction in the industry. On the basis of single factor experiment, the effects of liquid-to-material ratio, temperature, time, pH, and solid content on the ethanol extraction of tuna cooking liquid protein were studied by response surface test. Further, the structure of the extracted protein was characterized by Ultraviolet Scanning, Infrared Spectroscopy, Aamino Acid Analyzer and Circular Dichroism. The results showed that the optimal process conditions were temperature 36 ℃, time 2 h, liquid-to-material ratio 2.4, and the maximum protein extraction rate was 76.83%. The protein extracted by this method had the maximum absorption peak at about 218 nm, but no obvious absorption peak at 280 nm. The amino acid composition showed that the mass fraction of amino acids was 18.79%, and the content of hydroxyproline was 10.21%. The infrared spectrum had the characteristic absorption peak of gelatin. The above results indicate that the protein extracted from the cooking liquid by this process is gelatin.