The strategy of temperature downshift has been widely used in the biopharmaceutical industry to improve antibody production and cell-specific production rate (q p) with Chinese hamster ovary cells (CHO). However, the mechanism of temperature-induced metabolic rearrangement, especially important intracellular metabolic events, remains poorly understood. In this work, in order to explore the mechanisms of temperature-induced cell metabolism, we systematically assessed the differences in cell growth, antibody expression, and antibody quality between high-producing (HP) and low-producing (LP) CHO cell lines under both constant temperature (37°C) and temperature downshift (37°C→33°C) settings during fed-batch culture. Although the results showed that low-temperature culture during the late phase of exponential cell growth significantly reduced the maximum viable cell density (p<0.05) and induced cell cycle arrest in the G0/G1 phase, this temperature downshift leaded to a higher cellular viability, and increased antibody titer by 48% and 28% in HP and LP CHO cell cultures, respectively (p<0.001), and favored antibody quality reflected in reduced charge heterogeneity and molecular size heterogeneity. Combined extra- and intra-cellular metabolomics analyses revealed that temperature downshift significantly downregulated intracellular glycolytic and lipid metabolic pathways while upregulated TCA cycle, and particularly featured upregulated glutathione metabolic pathways. Interestingly, all these metabolic pathways were closely associated with the maintenance of intracellular redox state and oxidative stress-alleviating strategies. To experimentally address this, we developed two high-performance fluorescent biosensors, denoted SoNar and iNap1, for real-time monitoring of intracellular NAD +/NADH ratio and NADPH amount, respectively. Consistent with such metabolic rearrangements, the results showed that temperature downshift decreased the intracellular NAD +/NADH ratio, which might be ascribed to the re-consumption of lactate, and increased the intracellular NADPH amount (P<0.01) to scavenge intracellular reactive oxygen species (ROS) induced by the increased metabolic requirements for high-level expression of antibody. Collectively, this study provides a metabolic map of cellular metabolic rearrangement induced by temperature downshift and demonstrates the feasibility of real-time fluorescent biosensors for biological processes, thus potentially providing a new strategy for dynamic optimization of antibody production processes.