Lignocellulose is the most abundant renewable carbon source in the world. In the future, it is one of the important problems to be solved to develop clean energy with wood fiber as raw material. In order to investigate the effect of eukaryotes on the degradation of lignocellulose, by constructing the traditional PCR cloning library, cloning and sequencing combined with RDP fungi classification and identification, the composition of eukaryotic community structure and core population and diversity characteristics in wheat straw, corn stalks and wood decay samples were analyzed in the paper. The results showed that Opisthokonta（55%） and SAR（36.1%） were the most dominant species with the highest diversity, followed by Archaeplastida（0.3%） and undefined eukaryotes. The comparative analysis of three samples in the same period showed that the abundance and diversity of eukaryotes in wheat straw were significantly higher than that of corn stalks and wood decay samples, and the compositions and species diversity of eukaryotes during wheat straw decomposition were even higher. Different microorganisms carry different functions during the degradation of lignocellulose, and the study of these microorganisms will greatly enhance our utilization in lignocellulose degradation.