Although biological remediation of U-stressed soil has been studied for a long time, the combined effects of biochar and plant roots are rarely discussed and its influence on rhizosphere microecology are still unknown. Based on pot experiments, we explored the combined efforts of biochar addition and plant roots on U-stressed rhizosphere soil in several ways, including soil physicochemical properties, soil enzyme activities, uranium chemical speciation, bacterial community structure and metabolic pathways. Our results indicates that the content of DTPA-extractable U decreased by 49.31% after biochar and plant roots application, whereas plant roots only treatment just decreased by 25.46%. Further research has found that the pH, CEC, enzyme activities and nutritional level of rhizosphere soil were more significantly improved after biochar and plant roots application. Meanwhile, the abundance and diversity of bacterial community was also upregulated, which was also suggested by the stronger metabolisms of lipids, carbohydrate, nucleotides as well as amino acids. Correlation analyses also certified the positive associations between soil properties, bacterial communities and metabolism. We speculated that the uranium immobilization was mainly attributed to the direct fixation of biochar for its alkalinity, CEC, DOC, etc. and the joint action of biochar and plant roots for their stimulating effects on bacteria. Our findings suggested that combination of biochar and plant roots could limit bioaccessibility of U in a larger extend than plant roots only, which may be a better strategy for rapid remediation of U-streesed soil.