Lipase catalytic activity is greatly influenced by immobilization on nanoparticles. In this study, lipase from Aspergillus niger was immobilized on TiO 2 nanoparticles with different morphologies: microspheres, nanotubes, and nanosheets. All TiO 2 samples were prepared by a hydrothermal method. Lipase/TiO 2 nanocomposites were prepared by a physical adsorption method through hydrophobic interactions. The prepared composites were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and high-resolution transmission electron microscopy (HRTEM). The catalytic activity of free and immobilized lipases was tested using sunflower oil in the presence of methanol to produce biodiesel at 40 °C for 90 min. The lipase immobilized on TiO 2 microspheres showed the highest activity compared to the lipase immobilized on TiO 2 nanotubes and nanosheets. To optimize the lipase-to-microsphere ratio, lipase was immobilized on TiO 2 microspheres in different microspheres/lipase, w/w, (S/L) ratios of 1:1, 1:0.75, 1:0.5, and 1:0.25. It was noticed that the hydrolytic activity follows the order 1:0.25 > 1:0.5 > 1:75 > 1:1. The immobilization yield activities were found to be 113, 123, 125, and 130% for the microspheres/lipase (S/L) ratios of 1:1, 1:0.75, 1:0.5, and 1:0.25, respectively.