Abstract: In this study, three lipases—Rhizomucor miehei lipase, Thermomyces lanuginosa lipase, and Candida antarctica lipase B—were immobilized onto the silica support functionalized with 3-glycidyloxypropyl trimethoxysilane group through covalent attachment, achieving over 96% immobilization efficiency. The successful attachment of epoxy groups onto the silica surface was confirmed using Fourier-transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA). The thermal stability of the immobilized lipases enhanced significantly at elevated temperatures compared to their free counterparts, indicating that immobilization markedly improves enzyme thermal resilience. The catalytic performance of these immobilized enzymes was also evaluated in the biodiesel production process from soybean oil. The effect of factors such as temperature, enzyme loading, tert-butanol percentage, water content, and the presence of silica gel as a water adsorbent were investigated to optimize biodiesel yield. Results demonstrated that increasing the reaction temperature up to 50°C substantially improved the biodiesel production, establishing this as the optimal temperature. Additionally, the highest biodiesel yield was achieved when using 13% of the immobilized enzymes relative to the oil weight. Silica gel showed a positive effect on the biodiesel production process for all three immobilized lipase derivatives, with the highest yield observed at a 30% addition of this water absorbent. In the presence of 30% t-butanol, the biodiesel production yield using immobilized TLL and CAL B was completed successfully. However, this organic solvent did not serve as an effective modifier in the biodiesel production reaction using the immobilized RML. This study demonstrates that the use of immobilized enzymes on the functionalized silica support is an effective approach for producing sustainable and cost-effective biodiesel from soybean oil. It can serve as a significant step forward in developing green technologies and reducing environmental pollution.