Abstract <p>Waterlogging stress reduces the yield and quality of flue-cured tobacco (<i>Nicotiana tabacum</i> L.). At present, there is a lack of attention on the effects of waterlogging stress on the upper leaves of tobacco during the late-maturing stage. Here, we conducted pot experiments to analyze the changes of the physio-biochemical characteristics and the quality of tobacco leaves at the late-maturing stage on waterlogging conditions. The results showed that waterlogging decreased root vitality by 9.03 to 75.94%. Waterlogging stress significantly weakened the superoxide dismutase (SOD) activity, photosynthetic pigment content, and phototynthetic performance, while it significantly increased the malondialdehyde (MDA) content in tobacco leaves. The contents of total sugar, reducing sugar, nicotine, and potassium in tobacco leaves sharply decreased, whereas the total nitrogen content increased by 6.25–77.58%. The leaf color indexes, including lightness (L*&#xa0;value), redness (a* value), yellowish (b* value), saturation (c* value) and SPAD value were risen by waterlogging stress. Waterlogging for 12 h had minimal impact on physio-biochemical characteristics of tobacco roots and leaves, while significant changes were observed when waterlogging stress for more than 24 h. This study increases the knowledge of the effects of waterlogging stress on the physio-biochemical characteristics of upper leaves in tobacco at the late-maturing stage, as well as the evaluation of tobacco leaf quality, which provides reference for water management measures on tobacco at the late-maturing stage.</p>