Abstract: Ratooning cane monoculture leads to yield decline, but ratooning cane intercropped with legume crops increases the yields. The rhizosphere soils of “Ganzhe 18” newly planted sugarcane, ratooning sugarcane and its intercropping with soybean were used to study the alteration of the microflora and soil enzyme activities, and as well as some agronomic traits indexes. The sugar contents of the canes show no significant difference in different cultivation modes. However, owing to the larger diameter of ratooning cane, the ratooning cane intercropped with soybean has high yield. Furthermore, the soil enzyme activities in the rhizosphere soils under different cultivation modes are significantly different. The intercropping practice increases the enzyme activities, especially the activities of three soil enzymes involved in the cycles of carbon, nitrogen, and phosphorus, i.e., invertase, urease and phosphomonoesterase. The microbial biomass and the ratio of bacteria/fungi are decreased under the ratooning cane monoculture mode but recovered under the intercropping ratooning cane with soybean, suggesting a shift from fungal type to bacterial type in the compositions of microbial communities between two systems. The ratooning practice declines the total microbial biomass and the soil enzyme activities, suggesting that the nutrient cycles mediated by microbes in ratooning cane soil are blocked and thus decreased the yield of sugarcane, whereas, the intercropping mode has a significant improvement on soil microflora, which increases the yield of sugarcane.