During the usage of Rhizopus oryzae whole cell instead of immobilized enzyme for biodiesel production, the intracellular lipase was found to have 1,3-positional specificity when used to catalyze methanolysis of triglycerides. Thus, the application of R. oryzae whole cell in biodiesel production from triglycerides is restrained to some extent. However, it might be a promising catalyst for biodiesel production from free fatty acids (FFAs). In this paper, R. oryzae IFO4697 whole cell [immobilized within biomass support particles (BSPs)] catalyzed biodiesel production from oleic acid was studied systematically. It has been demonstrated that, in a tert-butanol system, R. oryzae whole cell exhibited both better methanol endurance and better stability than that in a solvent-free system. Molecular sieves (3 angstrom) were added into the reaction mixture to online remove the produced water, and a much higher biodiesel yield could be achieved (biodiesel yield reached 90% at 48 h).