A free living nitrogen-fixing bacteria called SGD 07 was selected and identified as Agrobacterium sp. based on its 16S rRNA gene sequence analysis. Atmospheric Plasma — Dielectric Barrier Discharge Plasma (DBD) was used for mutation of SGD07 and a mutant called MU7-3 was obtained, whose NA increased by about 60% than its original strain SGD 07. Furthermore,the Response Surface Methodology (RSM) was adopted to optimize the culture medium of MU 7-3 and two significant effects on the NA were selected by Planckett-Burman Design. Through the analysis of the results of full factorial central composite design, a quadratic polynomial model for NA was established. The final optimal values of the four variables were as follows:glucose concentration (19.92 g·L^-1), culture medium load (50ml), trace element ratio (0.014 ml·ml^-1), buffer solution concentration (0.015 mol·L^-1),  the maximal NA in optimized culture medium could reach up to 0.63 nmol·10⁷cfu^-1·h^-1, increased by 57.5% than the NA of 0.40 nmol·10⁷cfu^-1·h^-1 in the original culture medium. In general, the NA of MU7-3 increased about 152% via DBD mutation and optimization of culture medium by RSM compared to SGD 07 .