[Objective] To establish an efficient method for promoter characterization, the interaction between E. coli promoter and RNA polymerase (RNAp) was studied by atomic force microscope (AFM) based force spectroscopy.[Methods] Protein immobilization was optimized, and the specific promoter/RNAp interaction was verified using core-RNAp (RNAp-C) as control. The force spectrum of promoter Ls2, lack of -10 region of Ls1, was studied.[Results] Based on the established method, the specific interaction between promoter Ls1 and RNAp was studied, and the rupture force was measured as (331.10±5.14) pN. Ls2 showed significantly less binding events towards RNAp compared with Ls1. Using promoter-probe plasmid, the activities of Ls1 and Ls2 were verified by reporter gene-cat, which were (181.73±4.08) U/mg and (0.33±0.21) U/mg, respectively.[Conclusion] A novel promoter analysis method based on AFM force spectroscopy was established. The results demonstrated this method can be applied in quantitative characterization of promoter with high efficacy and reliability.