Abstract: To reveal the effects of different cultivation measures on TSNAs and their precursors in burley tobacco, an orthogonal experiment was conducted with different nitrogen application rates (210, 255 and 300 kg/ha), topping time (topping at buttoning stage, topping at budding stage and topping at initial flower stage) and BSM doses (0.1, 0.3 and 0.0 μmol/L). Contents of TSNAs, alkaloids and nitrate nitrogen were analyzed. The results showed that nitrogen application rate was the main influencing factor of the contents of TSNAs, alkaloids, nitrate-nitrogen and nitrite-nitrogen. Contents of TSNAs and their precursors of upper burley leaves were lower at 210 kg/ha nitrogen fertilizer. Topping time had an extremely significant effect on TSNA contents of upper leaves. When topping at budding stage TSNA contents were significantly lower than that of topping at buttoning stage and topping at initial flower stage. The contents of TSNAs, alkaloids, nitrate-nitrogen and nitrite-nitrogen with BSM treatment after topping were lower. At the conditions of this experiment, the optimal combination to reduce TSNAs is 210 kg/ha nitrogen application rate, topping at budding stage and 0.3 μmol/L BSM.