Abstract: Koji was prepared with Aspergillus oryzae as fermentation strain,with the whole wheat as raw material.The effects of fermentation parameters on protease activities were fully investigated,including the type of Aspergillus oryzae spores,the thickness of raw material,the initial moisture content of koji,relative humidity,fermentation temperature as well as culture time. The optimal koji-making conditions were as follows:the whole wheat was crushed to 20 mesh,the initial moisture of koji was 51%,the temperature was 32 ℃,and koji was obtained after 48 h under controlled condition(0~24 h,RH95%,24~36 h,RH90%,36~40 h,RH80%,40~44 h,RH70%). Under above conditions,the neutral protease activity and acid protease activity were as high as1583 U/g and 497 U/g,increased by 12.75% and 4.63% respectively compared with the previous study. The optimal koji-making parameters could provide experimental basis for the preparation of novel wheat flavored base material and seasonings on an industrial scale.