Cold stress-mediated reduced photosynthesis and osmotic stress severely endanger and limit plant development and crop yield. We investigated the expression of the NtPhyA (phytochrome A) gene in wild-type K326 and the defence response of PhyA knockout mutants under cold stress to monitor their physiological changes. PhyA mutants exhibited greater cold tolerance than wild-type (WT) plants, with lower levels of reactive oxygen species and malondialdehyde. They also had higher proline and antioxidant enzyme activities. The phytochrome signalling pathway regulated photosynthetic efficiency in tobacco leaves and maintained photosystem II activity during cold stress. NtPhyA responds to light and cold signals. It is regulated by hormones and transcription factors under stress. These results highlight the negative regulatory role of PhyA in the cold stress response of tobacco, and harnessing the function of the NtPhyA gene will improve cold tolerance, light and temperature insensitivity, and abiotic stress resistance in Nicotiana tabacum.