Taking wheat cultivars Yumai 34 (high protein content in grain) and Yangmai 9 (low protein content in grain) as test materials, and by the method of growth chamber, this paper studied the effects of different combinations of temperature and illumination on the photosynthetic characteristics and senescence of their flag leaves at grain-filling stage. The results showed that both high temperature and low illumination had negative effects on the photosynthesis. They decreased the net photosynthetic rate (Pn), photochemical efficiency (Fv/Fm), and actual photochemical efficiency (PhiPSII) significantly, but their action mechanisms were differed. High temperature mainly decreased the chlorophyll content (SPAD value) and Pn, while low illumination mainly decreased the chlorophyll fluorescence parameters Fv,/Fm and PhiPSII, and thus, inhibited the activity of PS II. High temperature increased the MDA content and decreased the soluble protein content and SOD activity, resulting in the acceleration of senescence, while low illumination increased the SOD activity and slowed down the senescence. Yumai 34 was more sensitive to high temperature and low illumination than Yangmai 9.