Aspergillus oryzae mycelium pellets with abundant aminoacylase were cultured by liquid fermentation. Mycelium pellets were immobilized by crosslinking method with reagent of gelatin and formaldehyde. Basing on the orthogonal design table L16 (4(5)), artificial neural network(ANN) model with back-propagation(BP) of error structured 4-10-15-1 was used to optimize the immobilization conditions. And the optimal conditions were obtained. Then the activity of the immobilized cells prepared under the optimal conditions was assayed. Compared with the free mycelium activity of 1800 u, the residual activity rate was 83%, which was in good accordance with that predicted by ANN. It illustrated that artificial neural network can be used to find optimal conditions for cell immobilization.