Utilizing whole cell biocatalyst for biodiesel production avoids the complex procedures of isolation, purification and immobilization of extracellular lipase. However, during repeated use of Rhizopus oryzae (R. oryzae) IF04697 whole cell for biodiesel production in solvent-free system, the whole cell exhibited very poor stability. The stability of cells has been found to be significantly improved after GA cross-linking. It seems that the lipase leakage was also reduced and the specific activity was maintained after cross-linking. Methyl esters could accumulate in cells and reduced the catalytic activity of cells while glycerol had no negative effect on lipase activity but impeded the mass transfer. Methyl ester was found to result in the change of secondary structure of lipase. GA cross-linking treatment improved the stability of R. oryzae IF04697 whole cell significantly and could retain the lipase structure. (C) 2010 Elsevier Ltd. All rights reserved.