Abstract: To investigate the effects of NtCEP7 peptide on tobacco growth, yield, and quality, this study screened the optimal application concentration of NtCEP7 peptide through pot experiments. Furthermore, field experiments were conducted to evaluate the effects of NtCEP7 foliar application, monitoring tobacco growth at different developmental stages and evaluating the quality and yield of flue-cured tobacco leaves. The results showed that foliar application of NtCEP7 peptide at all concentrations could promote the growth of tobacco plants. Considering both efficacy and cost, 1 μmol/L NtCEP7 was identified as the optimal concentration. Compared to the control group, the maximum leaf area, total plant mass, and aboveground mass of tobacco seedlings under 1 μmol/L NtCEP7 treatment increased by 11.10%, 31.62%, and 20.88%, respectively. Additionally, root application of NtCEP7 also promoted plant growth. Field experiments demonstrated that foliar application of 1 μmol/L NtCEP7 peptide at the seedling recovery stage, rosette stage and vigorous growth stage significantly improved the growth of tobacco plants. At the seedling recovery stage, the maximum leaf area, plant height, leaf number, and SPAD value of tobacco plants under NtCEP7 treatment increased by 79.44%, 58.36%, 36.83%, and 2.98%, respectively. At the rosette stage, the maximum leaf area and plant height were increased by 27.53% and 16.80%. At the vigorous growth stage, the maximum leaf area, plant height, stem circumference, and SPAD value increased by 15.60%, 5.09%, 13.52%, and 8.95%, respectively. Furthermore, NtCEP7 application significantly improved the appearance quality of flue-cured tobacco leaves, leading to a 4.85% increase in output value. Chemical analysis revealed that the nicotine and total nitrogen contents in the upper leaves were increased by 4.48% and 3.57%, and the reducing sugar and total sugar contents in the middle leaves were increased by 2.77% and 3.55%. Sensory evaluation showed that NtCEP7 treatment significantly improved the aroma and smoke characteristics of cured tobacco leaves, with notable improvements in the upper leaves. In conclusion, the NtCEP7 peptide can effectively promote tobacco growth, increase yield and improve quality, offering significant applied value in tobacco production.