To study the effects of secondary mutation on the ability for degradation of petroleum hydrocarbon and producing biosurfactant, microorganism from the oil contaminated soil was implanted again with low-energy N+ ions. Mutant strain 23A, obtained after the primary mutation with the low-energy N+ which could efficiently degrade oil and produce biosurfactant, was implanted again with low-energy N+. Secondary mutant strain degrades oil better and reduces the surface tension of culture medium significantly. Compared with the original strain 23A, mutant strain 23A-3 increases the degradation rate of total petroleum hydrocarbon by 17.3% and decreases the surface tension of culture medium by 25%. The results indicate that the secondary mutation has great potential to gain more effective microorganism. It has practical significance to improve the level of petroleum degradation further, and can enriche the technical route of ion beam bioengineering.Cited