Two N2-fixing isolates, B11S and B8S, were obtained from the surface-sterilized stalks of sugarcane variety GT11and RB86-7515 which were bred in Guangxi, China and introduced from Brazil, respectively. Both isolates showed acetylene reduction in nitrogen free media. Classification of both B11S and B8S was made on the basis of 16S rRNA sequence analysis, and the biological characteristics of the two isolates were studied. The 16S rRNA sequence of the isolate B11S had a 98.5% sequence similarity to those of many Stenotrophomonas maltophili and the 16S rRNA sequence of the isolate B8S had a 100% similarity to that of Agrobacterium tumefaciens (Rhizobium radiobacter). For both growth and nitrogenase activity of the isolates, the optimum temperature was about 31°C. The optimum pH was about 6.0 for their growth but 6.5~7.0 their nitrogenase activity. The two strains propagated more quickly and showed higher nitrogenase activity with the increase of carbon concentration in the culture media, and the effect of sucrose was better than that of the glucose. The growth and nitrogenase activity could be detected when the two strains were cultured in the media containing different concentration NH4+ and NO3-, and they grew better and showed higher nitrogenase activity under suitable nitrogen contents, but the growth of the two isolates was hindered more and more strongly with the increasing of the nitrogen content.