To understand the demand of water and nitrogen in tobacco seedlings during provisonal planting period, the effects of water and nitrogen interactions (with soil relative water content being 25%, 50% or 75% and nitrogen fertilizer applied at 0, 0.36, 0.72 g/kg ) on chlorophyll fluorescence characteristics in leaves of flue-cured tobacco seedlings were studied under artificial water control. The results showed that with the increase of soil relative water content, maximum quantum yield of PSⅡ(Fv/Fm), potential activation of PSⅡ(Fv/Fo), actual or effective quantum yield(ФPSⅡ), PSⅡ reaction center light trapping efficiency(Fv′/Fm′), photochemical quenching coefficient(qP) were decreased after an initial increase, while non photochemical quenching coefficient(qN) were constantly decreased. PSⅡreaction center photochemical activity reached optimal when the soil relative water content maintained at about 50%. Fv/Fm, Fv/Fo, Fv′/Fm′, ФPSⅡ and qP in leaves of tobacco seedlings were increased with increasing nitrogen application under different water treatment. Especially when the relative water content was 50% with nitrogen application at 0.36 g/kg, the PS Ⅱreaction center photochemical activity reached optimal. The effect of water on chlorophyll fluorescence parameters is more significantly than that of nitrogen application according to the results of gray relationship analysis. Cluster analysis results show that soil relative water content maintained at 50%, and nitrogen applied at 0.36 g/kg was the optimal treatment, followed by soil relative water content maintained at 50%, and nitrogen applied at 0.72 g/kg. This means that when soil relative water content maintained at about 50%, nitrogen application could effectively increase the PSⅡ function in leaves of flue-cured tobacco seedlings, but with the increase of nitrogen application, the promoting effect reduced gradually. In conclusion, the best agronomic measure for tobacco seedlings in provisonal planting period is soil relative water content maintained at about 50%, and nitrogen application at 0.36–0.72 g/kg.