Abstract: To address the issue of severe DNA degradation in tobacco leaves after baking, which hinders subsequent molecular identification experiments, this study comprehensively considered factors such as DNA concentration, quality, extraction efficiency, cost, and safety for molecular marker applications. Based on the SLS method, we conducted single-factor experiments and employed a Box-Behnken response surface model to determine the optimal DNA extraction protocol for cured tobacco leaves. Sample amount, centrifugation time, and water bath duration were used as experimental variables, with DNA concentration and the A₂₆₀/A₂₈₀ ratio as response values. The results showed that the optimal conditions for DNA extraction from cured tobacco leaves were 20 mg of sample, centrifugation 10 minutes of centrifugation, and 15 minutes in water bath. Under these conditions, the DNA concentration was 588.16±27.09 ng/µL, and the A₂₆₀/A₂₈₀ ratio was 1.75±0.03. These results were consistent with the predictions of Design Expert 11 software, confirming the reliability of the optimization method. Molecular marker applicability tests revealed that, compared to the pre-optimization protocol, DNA extracted using the optimized SLS method yielded clearer target fragment peaks in capillary electrophoresis after PCR amplification with SSR and KASP primers. Additionally, distinct genotyping plots were obtained using KASP markers, demonstrating its applicability for SSR and KASP molecular marker detection in flue-cured tobacco leaves of different varieties. Furthermore, the established molecular identification system combining the optimized SLS method with 13 selected SSR primer pairs effectively distinguished the tested cured tobacco varieties, demonstrating its applicability for cured tobacco cultivar identification.