In order to evaluate the high-value application of squid by-products yielded hydrolysate, the process of preparation and purification technology, chemical composition and in vitro antioxidant activity of the hydrolysate were investigated. The optimal conditions of papain hydrolysis were obtained by single-factor experiments and orthogonal test with the DPPH• scavenging ratio as index, amino acid composition was analysed by automatic amino acid analyzer, the hydrolysate was isolated with a Sephadex G-25 column. Based on single-factor experiments, the hydrolysate with the DPPH• scavenging ratio being 53.96 % was gained under the optimal condition of enzymolysis temperature of 45 °C, enzymolysis time of 3 h, total enzyme dose of 1.2 %, and the pH value of 7. The protein content of the hydrolysate reached up to 17.53 %, and the essential amino acids were accounted for 51.06 % of total amino acids. The largest content amino acid was glutamic acid, which accounted for 10.74 % of total amino acids. Compared with the amino acid profiles recommended by FAO/WHO, the quality of the protein hydrolysate was high, as it was rich in essential amino acids, including isoleucine, leucine, lysine, methionine and cystine, threonine, tryptophan, and valine, which covered 88 %-100 % of the FAO/WHO recommended. The hydrolysate was divided into three fractions (F1-F3) using a Sephadex G-25 column, the F1 possessed the highest antioxidation activity with the reducing power, •OH and DPPH• scavenging ratio being 0.236, 18.13 % and 63.85 % at the concentration of 5 mg/mL. Compared with the retention time of the reduced glutathione chromatomap, the relative molecular mass of F1 was higher than 307, F2 and F3 was lower than 307. The result revealed that the protein hydrolysate from squid by-products by papain had strongly antioxidant capacity in vitro and high nutrition, and this finding provided a new way of advanced exploitation of squid scrap resources.