Lipase synthesis was optimized using different substrates through solid-state fermentation followed by application of the produced lipase for biodiesel synthesis from multiple oils. Rhizopus oryzae RO-1 showed maximum lipase production and was selected for further use. The production of lipase by R. oryzae RO-1 could be improved when the substrate was supplemented with olive oil and the growth conditions were optimized. After partial purification, the enzyme was immobilized on four different supports. For the production of biodiesel, Amberlite-immobilized lipase was employed for transesterification of olive, canola, sunflower, soybean, and algal oil from Cladophora sp. The biodiesels produced were characterized by recording their Fourier transform infrared spectra. Biodiesel generated from algal oil contains high levels of polyunsaturated fatty acids, making it highly suitable as winter-grade biodiesel.