Boron (B) is a micronutrient tobacco needs in minute amounts, and Boron insufficient supply can causes significant tobacco yield loss, however, the appropriate concentration for flue-cured tobacco seedlings to growth remains unknown. In this sense, a hydroponic experiment was conduct to measure the agronomic traits, dry matter mass, chlorophyll content, photosynthetic performance, antioxidant enzymes, boron ion and nicotine content of flue-cured tobacco seedlings K326 under different boron concentrations of 0.000mmol/L (B1, CK), 0.125mmol/L (B2), 0.250mmol/L (B3), 0.750mmol/L (B4), 5.000mmol/L (B5), 10.000mmol/L (B6), 20.000mmol/L (B7), 40.000mmol/L (B8) after 30 days. B significantly influenced flue-tobacco seedlings growth on agronomic traits, photosynthetic performance, the activities of antioxidant enzymes, boron ion and nicotine content aspects. B linearly enhanced the accumulation of boron ion by 24.00%~96.44%, and decreased nicotine content by 21.60%~82.03% in tobacco seedlings. Solution B concentration at 0.750 and 5.000mmol/L markedly improved tobacco seedlings maximum leaf length by 4.83%~82.03% and leaf width by 0.77%~24.36%, root weight by 13.64%~56.82%, stem weight by 12.26%~52.36%, leaf weight by 9.68%~36.56%, dry matter mass by 10.65%~38.92%, the Pn parameter by 1.22%~80.28%, the Cond paramete by 33.40%~75.86%, while decreased the activities of SOD by 10.44%~91.67%, POD by 21.32%~65.62% and CAT by 50.05%~96.44%, and MDA by 16.23%~75.16%. The B concentration concluded in this study enhanced the agronomy traits, photosynthetic and biochemical characteristics of flue-cured tobacco seedlings, which lays a scientific theoretical foundation for rational application of B in tobacco production and improve the internal quality of flue-cured tobacco.