Abstract: Lipids in animals and plants exist in the form of lipid droplet (LD). It is very important to master the purification methods of lipid droplet and lipid droplet related protein (LDRP) for understanding the lipid changes and influence mechanisms of fish, and seeking appropriate regulatory approaches. In this paper, the lipid droplet and LDRP were separated and purified through centrifugation, resuspension and solubilization by using Oreochromis niloticus intestinal tissues as raw material. The results show that the intestinal adipose tissues were effectively removed by three times of centrifugation and three times of resuspension to obtain the pure lipid droplet. Although the concentration of LDRP protein extracted by solubilization was higher, the electrophoretic bands showed an obvious trailing phenomenon, indicating that the effect of lipid removal was poor, which affected the purity of LDRP extraction. Delipidation and solubilization need to be combined with four groups of organic solvents to obtain the LDRP extracted samples with high purity and good re-solubilization effects. The effects of centrifugation drying method and lipid droplet freezing on LDRP solubilization were not significantly different.