In the current study, biodegradation of benzene by free and immobilized bacteria using graphene oxide was studied under optimized conditions. Isolated benzene-degrading bacterium from contaminated soils was identified based on 16S rDNA gene sequencing and submitted to gene bank as Bacillus glycinifermentans Strain GO 13T. The optimum values of pH, temperature and benzene concentration for benzene degradation by free and immobilized cells were determined as 7, 32ºC and 1.25g/l, respectively using response surface methodology. Free cells were able to degrade 55.8% of Benzene after 24h under optimized conditions. Graphene oxide was used to immobilize Bacillus glycinifermentans Strain GO 13T. Analysis by FTIR and SEM showed that the strain adhered onto the graphene oxide surface and developed a biofilm. Immobilized cells were able to degrade up to 77% of benzene after 24h under optimized conditions. Results indicated that free and immobilized cells had a suitable application potential in the treatment of benzene-containing soils.