The winter wheat was selected to study the effect of biochar of cotton straw on root morphology and endophytic fungal communities by using the method of root scanning and Illumina Miseq high-throughput sequencing. The symbiosis mechanism was found by the test. The treatments were amount of 4.5t/hm2, 9.0t/hm2 and 13.5t/hm2 of straw biochar, respectively applied to the soil, and the straw returning was taken as test control in the field experiment. The results showed that root diameter and specific root length were increased significantly, and branching density and biomass were decreased significantly in the primary root, and only the root branching density was decreased in the secondary root, when the amount of biochar was 4.5 ~ 13.5t/hm2 in the mature period of winter wheat. At the fungal phyla level, the abundance of Ascomycota was increased significantly, and the abundances of Zygomycota, Chytridiomycota and unidentified fungus was reduced greatly, and the abundance of Glomeromycota was not significant. At the common fungal order level, the abundance of Pleosporales was increased significantly, and the abundance of Eurotiales and Mortierellales was reduced greatly. With the amount of 4.5~9.0t/hm2 biochar promoted the primary root growth of root branch density and biomass by decreasing the abundance of Eurotiales, and inhibited the secondary root growth of diameter and branch density by increasing the abundance of Pleosporales. By comprehensive comparison, the effect of amount of 9.0t/hm2 biochar on root morphology was better than that of other treatments. The treatment with amount of 9.0t/hm2 significantly increased the primary root diameter by 5.00% and specific root length by 33.57%, and decreased root branch density by 67.26% and biomass by 27.27%, while also reduced the secondary root diameter by 13.16% and branch density by 34.38%, compared with the test control at maturity stage of winter wheat.