Fish bone gelatin and fucoidan was used as the wall materials and multilayer fish oil emulsion was prepared by layer by layer self-assembly technology. Multilayer fish oil microcapsules were prepared by spray drying method and the storage stability was studied. According to the changes of Zeta potential, the optimum concentration of wall materials for multilayer emulsion was determined. On the basis of orthogonal experiment, the maximum microencapsulation efficiency of 82.03% was obtained when multi-layer fish oil microcapsules were prepared using the following optimum conditions: ultrasonic time 10 min, ultrasonic temperature 25 ℃, homogenization time 4 min, homogenization rate 5 000 r/min. The results of peroxide value showed that microencapsulation could significantly improve the storage stability of fish oil (P＜0.05), and the oxidation rate of three-layer fish oil microencapsulation was the lowest.