[Objective] To breed L-arginine high-yielding strains by atmospheric and room temperature plasma (ARTP), and to explore the optimal fermentation conditions of mutants by response surface method. [Methods] The L-homoarginine and 8-azaguanine resistant strain was obtained from the strain Corynebacterium glutamicum GUI089 (SGr, L-His?) that was treated by ARTP. Based on the results of single factor experiments, ammonium sulfate, glucose and urea were selected as independent variables to optimize fermentation conditions for L-arginine production from 7 factors by Plackett-Burman design. Subsequently, the levels of the three variables were further optimized by response surface methodology. [Results] After several rounds of screening, a strain was selected resistant to 15 g/L L-homoarginine and 0.7 g/L 8-azaguaine, and designated as C. glutamicum ARG 3-16 (L-HAr, 8-AZr, SGr, L-His?). L-arginine production of ARG 3-16 was 49.79% higher than that of the original strain. Beyond the higher production of L-arginine production, ARG 3-16 accumulated fewer by-products than the original strain, especially the accumulations of L-proline, L-glutamate and L-valine. Under the optimal conditions, the yield of L-arginine reached 39.72±0.75 g/L, 10.49% higher than before optimization. [Conclusion] We successfully bred a high-yielding L-arginine producing strain by ARTP breeding system, and optimized fermentation conditions by response surface methodology, the results suggested that ARG 3-16 was a promising L-arginine producing strain.