The pollution of polycyclic aromatic hydrocarbons was serious in sediments of the Pearl River estuary, China. A fluorene-degrading bacterium, strain A2-3, was isolated from hydrocarbon contaminated sediment of this estuary and identified as Rhodococcus sp. based on the analyses of 16S rRNA gene sequence and morphology. Rhodococcus sp. A2-3 can take naphthalene, p-Teropheny, fluorene, pyrene, salicylic acid, citric acid, acetic acid, diethyletheranhydrous, methanol or 4,4′-dibromodiphenyl ether as sole carbon source. 100% of 100 mg/L fluorene or 89% of 400 mg/L fluorene was removed in 7 days by strain A2-3 at 30 °C and pH 7.5. The strain A2-3 showed a high degradation efficiency of fluorene when pH values ranged from 5.5 to 8.5. The proposed pathway of fluorene catabolism by strain A2-3 was initially attacked by 3,4 dioxygenation. Our results suggested Rhodococcus sp. A2-3 can degrade PAHs under aerobic conditions and can function in bioremediation, particularly for weakly acid environment.