Green production of biodiesel via enzymatic transesterification necessitates technological improvements for practical implementation. Heterogenization of biocatalysts has long demonstrated its advantages in biocatalytic processes. In this work, a lipase from Thermomyces lanuginosus (TL) has been immobilized on biomimetic silica nanoparticles using two different strategies: in situ entrapment and adsorption/covalent surface immobilization. Variables such as mass of nanobiocatalyst, amount of immobilized enzyme, and activity per gram of silica were studied following a factorial design for the in situ immobilization. The influence of the enzymatic load on the stability and activity of the catalyst was studied for both the surface immobilized and the entrapped lipase. Immobilized preparations were characterized and assayed in the production of fatty acid methyl esters (FAMES). The entrapped nanobiocatalysts were more stable and active than the soluble TL, the commercial immobilized TL and the surface immobilized counterparts. However, surface immobilized lipase reached a maximum yield of 88% in the synthesis of FAMES from canola oil and methanol, surmounting from the yield obtained with the commercial immobilized TL by 10%. Accumulated specific productivity for the entrapped biocatalyst reached 65.6 μmoles FAME/g catalyst/min after repeated batch operation. Biomimetic silica demonstrated its versatility and robustness as a support for TL immobilization in the synthesis of FAMES.