Abstract: In order to understand the effects of plant growth regulators on the physiological and biochemical characteristics of the upper leaves of flue-cured tobacco, four growth regulators including chlorpyrifos, IAA, ethephon and ketamine were selected, and upper leaves of Yunyan 87 were used as the test materials. Agronomic traits, leaf structure, photosynthesis parameters, chlorophyll content, peroxidation index related enzymes, sugar and alkali content and sensory quality after baking were measured and analyzed. The results showed that spraying growth regulator had a significant effect on the opening condition, tissue structure and photosynthetic performance of the upper tobacco leaves. Among the growth regulators, clopidogrel had the best effect. Compared with CK, with the application of chlorpyrifos after topping, the area increased by 6.31%, the aspect ratio decreased by 3.17%, and the sea-gate ratio increased by 5.88%. At the same time, the net photosynthetic rate increased by 3.36%, and the chlorophyll content increased by 14.04%, which promoted the synthesis of carbohydrates, increased the ratio of sugar to alkali, and improved the post-curing sensory quality of upper leaves. In addition, chlorpromazine and IAA can significantly delay senescence of the upper leaves, while ethephon and ketamine promote senescence of the upper leaves. Taken together, the application of chlorpyrifos can improve the opening, tissue structure and sensory quality of the upper tobacco leaves, and improve the quality of the upper tobacco leaves.