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The embedded immobilized enzymes (<i>Rhizopus-oryzae</i>) on the magnetic nanoparticles (Fe₃O₄-NPs) is a new application for the sustainable production of high-quality biodiesel. In this study, biodiesel is derived from Kapok oil via ultrasonication (US)-assisted catalytic transesterification method. A novel attempt is made to prepare magnetic nanoparticles embedded by an immobilized enzyme to solve the problem of enzyme denaturation. This innovative method resulted in optimum biodiesel conversion of 89 ± 1.17% under reactant molar ratio (methanol: oil) of 6:1, catalyst loading 10 wt% with a reaction time of 4 h at 60 °C. The kinetic and thermal study reveals that conversion of Kapok oil to biodiesel follows a pseudo first-order reaction kinetic with a lower ΔE of 30.79 kJ mol⁻¹. The ΔH was found to be 28.06 kJ mol⁻¹ with a corresponding ΔS of −237.12 J mol⁻¹ K⁻¹ for Fatty Acid Methyl Ester formation. The ΔG was calculated to be from 102.28 to 109.40 kJ mol⁻¹ for temperature from 313 K to 343 K. The positive value of ΔH and ΔG is an indication of endothermic and non-spontaneous reaction. A negative ΔS indicates the reactant in the transition state possesses a higher degree of ordered geometry than in its ground state. The immobilized catalysts provided great advantages towards product separation and efficient biodiesel production. Highlights: 1. Effective catalytic transesterification assisted by the ultrasonication method was used for bi-odiesel production. 2. Magnetite nanoparticles synthesized by the co-precipitation method were used as heteroge-neous catalysts. 3. An immobilized enzyme (Rhizopus-oryzae) was embedded in the heterogeneous catalyst, as it is reusable and cost-effective. 4. The maximum biodiesel yield obtained from Kapok oil was 93 ± 1.04% by catalytic trans-esterification reactions.