Bacillus natto can convert sodium selenite into organic selenium. The growth curve of a strain of Bacillus natto was measured, and the optimum time and addition amounts of sodium selenite were determined. Using B.natto BSN424 as original strain, based on screening of Se-tolerance and Se-enri-ching, a genetic stable mutant strain BN-44 was obtained by atmospheric and room temperature plasma mutagenesis system. The results showed that the optimum time for selenium addition was at the 3rd hour in the culture period, and then the culture was continued to 24 hours. The optimum mass concentration of selenium in culture medium was 6μg/mL. The power of atmospheric and room temperature plasma mutagenesis system was 100W and the mutagenic time was 25s. After mutagenization, the BN-44 was eventually selected as the best strain with a high selenium content. Compared to the original strain, the selenium content after shaking and fermentation by BN-44 was increased from 742.12μg/g to 1136.43μg/g, which was increased by 53.13%. The results had shown that the atmospheric and room temperature plasma mutagenesis breeding method could mutagenize Bacillus natto BSN424 effectively, which provided a basis for the search of Se-enriching probiotic vector and its mutation breeding in organic selenium biotransformation.