Hunan is the main production area of double cropping rice in China. The extreme low temperature weather that may occur during the filling stage of late rice will cause rice yield reduction. In order to clarify the effect of increasing the application of biochar application on antioxidant enzyme activity and yield of late rice under low temperature at filling stage, a pot experiment was carried out with the rice variety Taiyou 390 and Xiangwanxian 1 as materials. The experiment set up three fertilization treatments and two temperature treatments at filling stage. Fertilization treatment includes conventional fertilization (NPK), conventional fertilization+increased application of biochar with 5% of soil weight (NPK+BC), and conventional fertilization+increased application of chemical fertilizer with the same amount of N, P, K contained in biochar (NPK+). The temperature treatments during the filling period are normal temperature (environment temperature) and low temperature (daily average temperature 17 ℃). The results showed that, compared with normal temperature at filling stage, the yield of Taiyou 390 and Xiangwanxian 1 decreased by 35.85% and 27.70% respectively at low temperature. The yield of the two rice varieties decreased in the order of NPK+BC<NPK<NPK+, and there were significant differences. Under low temperature stress at filling stage, the decrease of yield was very significantly negatively correlated with seed setting rate, but not significantly correlated with effective panicle number and grain number per panicle. Compared with normal temperature at filling stage, POD, CAT activity and MDA content in flag leaves and roots of Taiyou 390 and Xiangwanxian 1 increased under low temperature stress. At low temperature during filling stage, the activities of POD, SOD and CAT in flag leaves and roots of the two rice varieties were significantly higher in NPK+BC treatment than in NPK and NPK+ treatments. In conclusion, the low temperature stress at the filling stage led to the reduction of the yield of late rice, but the increasing the application of biochar could reduce the yield loss, which might be related to the increase of seed setting rate and antioxidant enzyme activity of late rice under low temperature stress at the filling stage.