Microbial biomineralization significantly affects the uranium (U) behavior in the environment. However, the mechanism of microbial biomineralization of U is still not fully understood. In this study, a dominant bacterium (Pseudomonas sp. WG2–6) was isolated from U tail mining area. Abiotic precipitation tests demonstrated that U biomineralization was entirely attributed to the mediation of Pseudomonas sp. WG2–6 when the concentration ratio of exogenous β-glycerophosphate (SGP) to U was 10:1. Pseudomonas sp. WG2–6 exhibited strong immobilization ability towards U (97.59 %) according to batch experiments, and acylamide, carbonyls, and phosphate groups were the main functional groups that interacted with U. Besides, U mainly existed in the form of amorphous U-P complexes after biomineralization by Pseudomonas WG2–6, which could be converted into crystalline nano-minerals H2(UO2)2(PO4)2·8H2O in the presence of SGP. In particular, the formation and structural composition changes of extracellular polymeric substance (EPS) as well as the decrease in U4f binding energy were observed during the U biomineralization process of Pseudomonas sp. WG2–6 in the presence of SGP, indicating that EPS provided the nucleation site for the formation of stable biomineralized products. This work provides new insight into the mechanism of U microbial biomineralization and a theoretical basis for the remediation of U contaminated environments through microbial biomineralization.