The bacterial strain MJ01 was isolated from stock tank water of one of the Iranian south oil field production facilities. The 16S rRNA gene of isolate, MJ01, showed 99% similarity to Bacillus subtilis. The results revealed that biosurfactant produced by this strain was lipopeptide-like surfactin based on FTIR analysis. Critical micelle concentration of produced surfactin in distilled water was 0.06 g/l. Wettability study showed that at zero salinity surfactin can change original oil-wet state to water-wet state, but in seawater salinity it cannot modify the wettability significantly. To utilize this biosurfactant in ex situ MEOR process, economical and reservoir engineering technical parameters were considered to introduce a new optimization strategy using the response surface methodology. Comparing the result of this optimization strategy with the previous optimization research works was shown that significant save in use of nutrients is possible by using this medium. Furthermore, using this method leads to less formation damage due to the incompatibility of injecting fluid and formation brine, and less formation damage due to the bioplugging.