As a natural cationic polymer, chitosan has good biocompatibility and is one of the ideal wall materials of microcapsules, while fish oil is oxidizable material. Low temperature is beneficial to the processing of fish oil. Freeze-drying technology can alleviate the oxidation of fish oil. Therefore in this experiment, microcapsules of fish oil are made by freeze-drying method, with chitosan as wall material and tuna oil as core material, and response surface analysis is used to optimize the preparation process parameters. Results show that, with the encapsulation efficiency as the evaluation index, according to the design principle of Box-Behnken center combined test based on single factor test, and in combination with the actual production situation, the optimal preparation process parameters of microcapsules are confirmed to be 2.1% of wall material concentration, 1.9:1 of wall-core ratio, 2:1 of the ratio of emulsifiers Tween80 to Span80, and 0.9% of emulsifier content. Under such conditions, the theoretical value of encapsulation efficiency is 79.67%, while the actual average value is 79.45%. The physical and chemical indexes of microcapsules made are up to industrial standards. The results of this study not only can provide a practical reference for preparation of microcapsules of fish oil by freeze-drying method, but also provide an experimental theoretical basis for the preparation of chitosan microcapsules.