Atomic force microscopy (AFM) was employed to quantitatively detect the interaction between a transcription factor, ZmDREB1A, and its target DNA sequence (DRE).The specificity of the interaction was revealed by comparing the binding probability of ZmDREB1A to DRE element with core sequence ACCGAC and to the ERE element with core sequence AGCCGCC. Moreover, the single-molecular unbinding force between ZmDREB1A and DRE element was determined using Poisson statistical method to be 99 ± 9 pN.The results indicated that AFM could serve as a sensitive and reliable technique to probe the interaction properties of the transcription factor and DNA element at single-molecular level, thus indicating that AFM is advantageous over the traditional methods, such as the electrophoretic mobility shift assay, regarding to the functional analysis of transcription factors.