The unreasonable application of chemical fertilizer as abiotic stress can affect the production of reactive oxygen species (ROS) and the synthesis of related substances, thereby affecting the antioxidant systems mediated by Ascorbate-glutathione (AsA-GSH) cycle, Methyl jasmonate (MeJA), S-Nitrosothiols (SNOs) in tobacco plants. Up to now, further research is needed on the impact of chemical fertilizer application on the above antioxidant system in tobacco plants. In this study, the flue-cured tobacco cultivar ‘Yunyan 87’ (Nicotiana tabacum L.) was used as the material. The content of non-enzyme antioxidants and related enzyme activities of AsA-GSH cycle system, the content of MeJA and SNOs in tobacco leaves, as well as tobacco yield were detected, to study the effects of different amount of fertilizer application (T1 = 30 g/plant, T2 = 45 g/plant, T3 = 60 g/plant) on related indicators of antioxidant system. The results indicated that the content of non-enzymatic antioxidants (AsA and GSH) and the activities of related enzymes in the AsA-GSH cycle, including ascorbate peroxidase (APX), glutathione reductase (GR) and monodehydroascorbate reductase (MDHAR) exhibited first increased and then decreased with increasing amount of fertilizer application. Most of the treatments showed significant differences. T3 treatment markedly reduced MeJA content compared to T1 and T2 treatments. With the increase of fertilizer application, the content of SNOs and the activity of S-Nitrosoglutathione reductase (GSNOR) in tobacco leaves exhibited an initial increase, followed by a decrease, and finally increasing trend, with notable differences among most treatments. Tobacco yield markedly decreased in the T1 treatment compared to the T2 and T3 treatments; however, no obvious difference between T2 and T3 treatments. Altogether, the fertilizer application of T1 (30 g/plant) was too low, which was not conducive to the synthesis of antioxidant substances in tobacco leaves. However, the fertilizer application amount of T3 (60 g/plant) was attributed to excessive application, which caused high salt stress on the growth of tobacco plants. This study provides a basic reference for optimizing fertilizer application in tobacco production.