Abstract: In this research, flue-cured tobacco cultivar (Nicotiana tabacum L. K 326) was selected to study the effect of soil-N on nitrogen accumulation and nicotine biosynthesis in flue-cured tobacco using the N15-trace method in a field experiment. The results indicated that soil-N ratios had an increasing effect on nitrogen accumulation and nicotine synthesis with leaf position ascending, and the effect of soil-N ratios of three leaf positions and root and stem after removing the apex also increased with growth stages. Soil-N ratios of all organs after removing the apex were higher 50% and 60%, all significantly higher than fertilizer-N ratios.