To explore an efficient nutrient co-application fertilization mode promoting the accumulation of sweetness-related substances in tobacco leaves, a field experiment was conducted under two nitrogen (N) application levels (conventional N and increased N application) with three N-to-P ratios (1:0.7, 1:1, and 1:1.3), and the effects of different N-P ratios on tobacco growth, photosynthesis, and the activities of key enzymes involved in sugar synthesis within carbon metabolism were studied. The results showed that under the same N-P ratio, increased N application promoted plant height and stem girth at the rosette stage. At the vigorous stage, it conferred no significant gain in most agronomic traits or photosynthetic indicators, except for a significant increase in leaf width. Nevertheless, under the same N level, increasing P application significantly enhanced the length and width of the largest leaf, as well as the intercellular CO₂ concentration, photosynthetic rate, and stomatal conductance of the tobacco leaves. Under conventional N application, increased P application elevated the activity of sucrose phosphate synthase in the leaves but had no effect on the activity of ADP-glucose pyrophosphorylase (AGPase), a key rate-limiting enzyme in starch synthesis. Increased N application inhibited sucrose accumulation, while reducing the N:P ratio by adding P alleviated this negative effect and promoted total sugar accumulation. In conclusion, in the tobacco-rice rotation system of southern Hunan, appropriately increasing P application to reduce the N:P ratio while controlling nitrogen input can improve agronomic traits, enhance photosynthetic capacity and carbon metabolism activity, and thereby improving tobacco leaf quality.