Abstract: To clarify the effects of different harvesting maturity levels on the quality formation of upper leaves of flue-cured tobacco, the main cultivar “Zhongyan 100” grown in Xuchang was used as the experimental material. Field experiments were conducted with different harvesting maturity treatments. SPAD value, key enzymes related to carbon and nitrogen metabolism, as well as conventional chemical components, aroma compounds, appearance quality, and sensory quality of flue-cured tobacco leaves were determined to systematically analyze the effects of harvesting maturity on tobacco leaf quality. The results showed that with the increase of harvesting maturity, the SPAD value of fresh leaves gradually decreased. Amylase (AMY) activity exhibited a trend of first increasing and then decreasing, while the activities of invertase (INV), nitrate reductase (NR), and glutamine synthetase (GS) showed an overall decreasing trend. Appropriate delay in harvesting promoted starch degradation, increased the contents of total sugar and reducing sugar in cured leaves, and enhanced the accumulation of aroma-related compounds such as degradation products of aromatic amino acids and carotenoids, thereby improving tobacco quality. T₃ treatment showed superior performance, with higher total sugar and reducing sugar contents, greater accumulation of aroma substances, appropriate nicotine content, and the highest scores in appearance and sensory evaluation. The proportion of high-grade tobacco in T₃ increased by 22.00%, 8.88% and 18.28% compared with T₁, T₂, and T₄, respectively. Other treatments, due to insufficient or excessive maturity, were not conducive to tobacco quality formation. In conclusion, appropriately delaying the harvesting time of upper tobacco leaves can enhance maturity, promote carbon and nitrogen metabolism transformation and aroma compound accumulation, and improve the coordination of chemical composition and sensory quality. Among the treatments, T₃ performed the best and can be considered as the optimal harvesting maturity upper for leaves of flue-cured tobacco.