Abstract: In this research, Pinctada martensiiwas was used as the raw material, as its oligosaccharide was obtained by simulating the conditions of gastrointestinal tract digestive system. After hydrolyzed with pepsin, the hydrolysis process was optimized under the combined action of trypsin and chymotrypsin. According to the analysis of artificial neural networks, the result demonstrated that enzyme-to-substrate level of 4 500 U/g (trypsin/chymotrypsin ratio of 61), substrate-to-liquid ratio of 12 and reaction time of 3.5 h were the optimal conditions to generate the oligosaccharide (200-2 000 u MW). Under these conditions, a relatively high yield of P. martensii oligosaccharide could be obtained, which would lay a foundation for its further exploration and utilization.