Although sugarcane-soybean intercropping has been widely used to control disease and improve productivity in the field, the response of soil fungal communities to intercropping has not been fully understood. In this study, the rhizosphere fungal communities of sugarcane and soybean under monoculture and intercropping systems were investigated using Illumina MiSeq sequencing of ITS gene. Intercropping decreased the alpha-diversity and changed fungal community composition compared to monocultures. Taxonomic analyses showed that the dominant phyla were Ascomycota, Zygomycota and Basidiomycota. The abundance of Ascomycota decreased in intercropping sugarcane-grown soil compared to monoculture, while it increased in soybean-grown soil in the intercropping system. In addition, intercropping increased the abundance of important fungal genera, such as Trichoderma, Hypocreales and Fusarium but decreased the relative abundance of Gibberella and Chaetomium. The results of canonical correspondence analysis and automatic linear modelling indicated that fungal community compositions were closely associated with soil parameters such as total nitrogen (TN), soil organic matter (SOC), pH and NO3-, which suggests that the impacts of intercropping on the soil fungal community are linked to the alteration of soil chemical properties.