A method for preparing microbial oils from wood fiber materials, which belongs to the field of biochemical technology, comprises the following steps: preparing hydrolysate from wood fiber materials by two hydrolysis steps, and preparing the microbial oils from the hydrolysate by two material feeding and fermenting processes. The method can prepare microbial oils with high output and content from the wood fiber materials. The fermentation substrate and the feeding substrate can be selected from the hydrolysate of the wood fiber materials, thus sufficiently utilizing the wood fiber resources and lowering the production cost.