The microorganism trench-leaching process of a low-grade uranium ore with a high content of fluorine was experimentally studied through the technique of Real-time PCR by using the fluorine-tolerance bacteria of Retech-3 as the leaching-ore bacteria. It was found that the Retech-3 strains of fluorine-tolerance domestication grown well in a fluoride solution with concentration of 0.0~3.0 g·L^-1, and the oxidation of Fe²⁺ ions to Fe³⁺ ions occurred rapidly to promote the extraction of uranium. The experimental results showed that the bioleaching rate (89.82%) of uranium can be improved of 13.80% than acid leaching in 30 days with 65 kg ore and its particle size of-6 mm (fraction of 100%) in trench. The Real-time PCR analysis of microbial population compositions revealed that Retech-3 strains were mainly composed of Leptospirillum sp. and Acidithiobacillus sp. in the leaching process. The obvious improvements of fluorine-tolerance domestication of bacteria and the uranium leaching process will provide fundamentals for the smooth utilization of a high fluorine-containing uranium ore with the aid of microorganism trench-leaching.