Abstract: In this study, κ-carrageenan oligosaccharides were prepared by enzymatic hyrolysis method to improve the technology.Hydrolysis conditions were optimized with the characteristic signs of reducing sugars content, and the optimal conditions for κ-carrageenan oligosaccharides preparation were obtained as substrate concentration 12 g/L, p H7.0, reaction temperature 40 ℃ and shaking speed 100 r/min, respectively.Compared with the reducing sugars yield of the initial conditions, the yield of the optimal conditions increased from 0.91 mg/m L to 1.61 mg/m L, which was increased by 77%.The Kmand Vmax values of the enzyme hydrolysis reaction was 171.96 mg/m L and 0.925 mg·m L^-1·min^-1, respectively. Then, the result of optimal process validation test could consistent with that of 20 L enlarge experiment.At last, the enzymatic hydrolysis products were analyzed by thin-layer chromatography and mass spectrometry, the result showed that the main products included tetrasaccharide and hexasaccharides in 24 h reaction.