This study was conducted to investigate effects of glyphosate stress on physiological characteristics and photosystem Ⅱ (PSII) protein level in genetically modified soybean GTS 40-3-2 seedlings under severe drought condition. To achieve this objective, a pot experiment was carried out in growth chamber to determine the response of V3 stage of genetically modified soybean treated with severe drought stress and different concentrations of glyphosate. Severe drought treatment increased the Electrolyte leakage(EL), Superoxide dismutase (SOD) and Peroxidase (POD) activities, and decreased relative water content(RWC), chlorophyll content, and Catalase (CAT) activity. The EL, SOD and POD activities were significantly increased under severe drought and glyphosate treatments, which were related to glyphosate concentrations. The chlorophyll content was decreased, which was also related to glyphosate concentrations. But the RWC and CAT activity were not affected by glyphosate concentrations. Western blot displayed PSⅡ protein Lhcb2 was not affected by stress conditions and stably expressed. D1, D2 and Lhcb4 protein level decreased, and there was no significant change of Lhcb1 expression under severe drought stress. The protein level of D1, D2, Lhcb1 and Lhcb4 decreased with glyphosate concentrations under severe drought and glyphosate stress. When the glyphosate concentrations were 0.92 and 1.84 kg.ai.hm-2, the protein level of D1, D2 and Lhcb4 were slightly higher than those in severe drought stress. When the glyphosate concentrations were 3.68 and 7.36 kg.ai.hm-2, the protein level of D1, D2, Lhcb1 and Lhcb4 decreased sharply. This research provides theoretical basis for cultivation of genetically modified soybean.