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Five strain of desulfurizing bacteria were isolated using DBT as a sulfur source. Among these strains, R. gordoniae R3 was able to endure a high concentration of substrate due to its ability to transform the toxic substance into non-toxic form. This strain also shows a high DBT desulfurization rate as compared to the well characterized strain, R. erythropolis IGTS8. The disadvantage of this strain is the breaking of the ring structure of DBT during desulfurization process. This strain was selected for cloning of sulfurspecific desulfurization gene. Even though the sulfur-specific pathway of the recombinant R. gordoniae R3 was not achieved after tested by Gibb's assay test, the difference of protein synthesis could be observed by SDS-PAGE. Its ability to transform DBT into non-toxic form was also reveal by GC-MS analysis of fatty acids.