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Whole cells of Aspergillus oryzae 35 were used as a biocatalyst for the degradation of glycerol trioleate, which largely contributes to pitch deposits in papermaking. Different types of inducers in culture media showed various effects on both the biomass and lipase activity of whole-cell biocatalysts. The cells of A. oryzae 35 cultured with Tween 80 showed higher catalytic activity than the others. The effects of several key factors on A. oryzae 35-catalyzed treatment of a glycerol trioleate-containing whitewater model were investigated, and the optimal pH value, reaction temperature, substrate concentration, and shaking speed were determined to be 7.5, 45 °C, 50%, and 200 rpm, respectively. Results from a practical treatment of whitewater resin sediments with A. oryzae showed that the mean particle size of the original whitewater was sharply reduced from 534 nm to 356 nm after biocatalytic degradation. Aspergillus oryzae whole-cells are newly promising biocatalysts for whitewater treatment in papermaking industries because of their cost-effectiveness, simple preparation, and environmental friendliness.