The addition of soil amendments to facilitate plantbased remediation of soil contaminated with radioactive nuclides is considered a promising approach. Here, we tested different levels of biochar to help clean uranium-contaminated soil in the potted plants. Adding 1% biochar had the best results in deactivating uranium, increasing soil enzyme activity, and promoting ryegrass growth. Microbiological and metabolomic analysis further revealed that 1 wt % biochar significantly enhanced the abundance of microorganisms such as Actinobacteriota and Myxococcota and accelerated the production of differential metabolites such as lipids and lipid-like molecules, organic acids and derivatives, and organic oxygen compounds. The analysis of biological and nonbiological interaction networks indicates that the coordinated interaction between bacteria, enzymes, and metabolites significantly improves the expression level of the ABC transporter's metabolic pathway. This enhances the resistance of living cells to uranium and maintains system homeostasis under uranium stress. This study provides an example of the application of biochar-assisted phytoremediation and offers theoretical guidance for the remediation of soil contaminated with radioactive nuclides.