Using lignosterol and oleic acid as raw materials and Candida rugosa lipase covalently immobilized on magnetic chitosan nanocomposites as catalyst, the conditions for enzymatic synthesis of lignosterol oleate were systematically optimized. Under the optimized conditions of catalyst dosage 12.7% (based on the total mass of substrates), substrate concentration of lignosterol 121.5 g/L, molar ratio of oleic acid to lignosterol 2:1, reaction temperature 50°C, reaction time 24 h, the conversion rate of lignosterol could reach 96.42%. For lauric acid, myristic acid, palmitic acid and mixed fatty acids composed of fatty acids with different chain lengths, the conversion rate of lignosterol could still be as high as 96.67%~98.74%. The immobilized biocatalyst showed good reusability. After being reused for 5 times, the conversion rate of lignosterol could still be obtained in 82.45%.