Photosynthetic dysfunction and reactive oxygen species (ROS) production are the common features of plant stress responses. Based on quantitative measurement of ROS production and delayed fluorescence (DF) emission, which is an excellent marker for evaluating photosynthesis, an on-line multiparameter analyzing optical biosensor for detecting plant stress responses was developed. Performances of the proposed biosensor were tested in the wild type (WT) Arabidopsis and heat shock protein (Hsp) 101 T-DNA knockout mutant (hsp101) plants with different thermotolerance. Results demonstrated that DF intensity correlates with net photosynthesis rate (Pn) in response to elevated temperature in both the WT Arabidopsis and hsp101 mutant plants. The light response characteristics and the recovery dynamics of the DF intensity were also in line with those of Pn in both the WT Arabidopsis and hsp101 mutant plants after heat stress (HS, 40℃ for 30 min), respectively. In all experiments discussed above, the hsp101 plant showed the worse photosynthetic performance than the WT plant. Moreover, after HS, more ROS production in the hsp101 mutant than in WT Arabidopsis, which was found to be mainly localized at chloroplasts, could be directly detected by using the proposed biosensor. In addition, the hsp101 mutant showed severer chloroplasts alterations than the WT plant within the first 1 h of recovery following HS. Nevertheless, pre-infiltration with catalase (CAT) reduced ROS production and prevented the declines of the DF intensity. Therefore, HS-caused declines of photosynthetic performance might be due to oxidative damage to photosynthetic organelle. To sum up, we conclude that Hsp101 plays an important role in preventing oxidative stress, and the proposed optical biosensor might be a powerful tool to determine plant stress responses and identify plant resistant difference. Photosynthetic dysfunction and reactive oxygen species (ROS) production are the common features of plant stress responses. Based on quantitative measurement of ROS production and delayed fluorescence (DF) emission, which is an excellent marker for analyzing photosynthesis, an on-line multiparameter analyzing optical Biosensor for detecting plant stress responses was developed. Performances of the proposed biosensor were tested in the wild type (WT) Arabidopsis and heat shock protein (Hsp) 101 T-DNA knockout mutant (hsp101) plants with different thermotolerance. correlates with net photosynthesis rate (Pn) in response to elevated temperature in both the WT Arabidopsis and hsp101 mutant plants. The light response characteristics and the recovery dynamics of the DF intensity were also in line with those of Pn in both WT Arabidopsis and hsp101 mutant plants after heat stress (HS, 40 ° C for 30 min), respectively , the hsp101 plant showed the worse photosynthetic performance than the WT plant. Moreover, after HS, more ROS production in the hsp101 mutant than in WT Arabidopsis, which was found to be mainly localized at chloroplasts, could be directly detected by using the proposed biosensor . In addition, the hsp101 mutant showed severer chloroplasts alterations than the WT plant within the first 1 h of recovery the following HS. Nevertheless, pre-infiltration with catalase (CAT) reduced ROS production and prevented the declines of the DF intensity. Therefore, HS -caused declines of photosynthetic performance might be due to oxidative damage to photosynthetic organelle. To sum up, we conclude that Hsp101 plays an important role in preventing oxidative stress, and the proposed optical biosensor might be a powerful tool to determine plant stress responses and identify plant resistant difference.