Alzheimer's disease(AD) is a neurodegenerative disease with memory decline, cognitive decline, language and psychomotor disorders as the main clinical features. With the aging of the global population and the deterioration of living environment, the number of patients is also increasing. According to the World Alzheimer's Disease Report, the global population of Alzheimer's patients is expected to grow to 152 million by 2050, it will have a huge impact on the world economy and society. The cholinergic hypothesis was one of the first and still widely accepted mechanisms to describe the pathology of AD, which considered acetylcholine as an important neurotransmitter involved in learning and memory. The lack of acetylcholine will lead to insufficient cholinergic signal transmission, which will lead to AD. Acetylcholinesterase(AChE) inhibitors can reduce the decomposition of acetylcholine by inhibiting the activity of AChE, which makes them one of the main drugs for the treatment of AD. The AChE inhibitors drugs approved by FDA include Donepezil, galantamine, etc. However, the micro-molecule drugs have strong side effects, so it is necessary to find new safe and efficient AChE inhibitors. Such as pathological protein aggregation, oxidative stress and metal ion homeostasis imbalance will aggravate the disease process, and there is a correlation between different pathogenic factors, which promotes the change of drug research direction from single target to multiple targets. Bioactive peptides are peptide compounds with biological activity, which have the advantages of high selectivity, high specificity, multi-target, high safety and low immunogenicity. In particular, bioactive peptides from Marine sources have many specific structures and functions due to their unique growth environment. Researchers have prepared and isolated peptides from Marine organisms with antioxidant, anti-inflammatory, blood pressure, uric acid, immune regulation and other effects. Trachinotus ovatus has the advantages of high yield, fast growth, strong disease resistance, high nutritional value, high protein and essential amino acid content, while it is a good raw material for preparing bioactive peptides. Enzymatic hydrolysis is the most commonly used method for preparing peptides, which is mild, controllable and low cost. In this study, the AChE inhibitory peptide was prepared by enzymolysis of Trachinotus ovatus. The optimal hydrolysis protease and enzymolysis time were selected based on AChE inhibition activity. The effects of hydrolysis degree, molecular weight distribution, amino acid composition and other enzymolysis characteristics on bioactivity were analyzed. The results showed that the 4 h hydrolysate of papain had the highest inhibitory activity of AChE, the inhibitory rate was 18.02±0.78%, which was much higher than that of positive control cerebrolin 2.05±0.45%. At the same time, the antioxidant activity of the enzymolysis was the best, and the ABTS radical scavenging capacity was 52.54±0.89%. The correlation analysis of AChE inhibition activity and ABTS radical scavenging capacity at different time showed a high positive correlation, and the correlation coefficient was 0.845(P<0.01). The result might be caused by the production of more small molecular weight peptides and the exposure of amino acid residues by enzymolysis. The physicochemical indexes of the enzymolysis products were further determined，the results showed that the protein content was 88.39%, the degree of hydrolysis was 14.86%, the components with molecular weight < 3 000 Da accounted for 96.87%, the hydrophobic amino acids accounted for 34.92%, and the contents of glutamic acid, aspartate, lysine and leucine were high. Low molecular weight and hydrophobicity transported into the membrane through passive will facilitate the passage of the enzymolysis across the blood-brain barrier. In addition, the enzymatic hydrolysis product can bind Ca2+ and Fe2+ with binding rates of 26.28% and 14.25%, respectively, which can regulate the balance of metal ions in the body by excessive calcium and iron ions in the chelate. And the AChE inhibitory activity is improved after binding, which may be caused by changes in spatial and electronic structure after binding. Ultraviolet analysis showed that the hydrolysates interacted with calcium and iron to form new compounds, and Fourier transform infrared spectroscopy showed that amino and carboxyl groups participated in the formation of the complexes. Based on the above results, the screened hydrolysate has good potential for treating AD. In the future, we will further verify the AChE inhibition effect of AChE inhibitory peptide in vivo, explore the main role of peptide composition, explore the mechanism of action. This will provide theoretical support and scientific basis for Marine bioactive peptides in the improvement and treatment of AD disease.