Abstract: This study employed nano-SiO₂ modified with trimethoxyphenylsilane (TMPS) and trimethoxyoctylsilane (TMOS) as carriers for the immobilization of Rhizopus oryzae lipase (ROL). The effects of the differently modified carriers on the enzymatic alcoholysis of fish oil for enriching eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were investigated. By measuring enzyme activity and analyzing reaction products, the loading efficiency of the two immobilized enzymes (ROL@SiO₂-TMPS and ROL@SiO₂-TMOS) and their enrichment effects on EPA and DHA were compared. The results show that TMOS provides superior hydrophobic modification of SiO₂, significantly enhancing enzyme loading (92.08%). During the alcoholysis reaction, ROL@SiO₂-TMPS exhibites better enrichment for EPA (EPA content reaches 1.93 times the initial level at 2 h, with a recovery rate of 98.62%), while ROL@SiO₂-TMOS showes better enrichment for DHA (DHA content reaches 1.98 times the initial level at 2 h, with a recovery rate of 98.25%). The enriched EPA and DHA are primarily distributed in diglycerides. Simultaneously, the by-product fatty acid ethyl esters (FAEEs) generated in the reaction were rich in myristic acid, palmitic acid, and palmitoleic acid (sum >80%), which can be utilized as biodiesel. The findings of this study achieves efficient enrichment of EPA and DHA coupled with high-value utilization of by-products, providing an effective approach for the comprehensive utilization of fish oil resources.