The invention provides a cellulase preparation method and application. The preparation method includes: subjecting trichoderma reesei Rut-C30 to standing fermentation for 24-72h, performing inoculation of penicillium oxalicum 16, and continuing fermentation for 3-10d to obtain cellulase. A preparation method of a cellulase culture medium includes: well mixing agricultural wastes with nutrient solution according to a mass ratio of 1:1-5, controlling the temperature at 100-145 DEG C, and sterilizing for 0.5-2h to obtain the cellulase culture medium, wherein the agricultural wastes include strawand bran, and the nutrient solution comprises KH2PO4, (NH4)2SO4, urea, MgSO4, CaCl2 and Mendel salt solution. By adoption of a response surface method, predicated values of any test points in a test range are determined, response values are controlled according to a mathematical model, and different operating parameters are selected to seek optimal process parameters. By the cellulase preparationmethod and application, a quick and applicable technique is provided for eukaryotic microorganism based high-level production of lignocellulose enzymes for alcohol fermentation, and a scientific basisis provided for further research on agricultural waste based ethyl alcohol production.