TSH1, a novel transcription factor in rice containing conserved AP2/EREBP domain, was screened from the EST database. The specific interaction of TSH1 and DNA elements was detected by two kinds of traditional methods, electronic mobility assay and yeast one-hybrid assay, which suggested that THS1 specifically binds to GCC element in vitro and in vivo. Furthermore, It was confirmed by atom force microscopy (AFM) that TSH1 binds to GCC element (core sequence GCCGCC) not DRE element (core sequence GCCGAC), and the single molecular force between TSH1 and GCC element was quantitatively measured. The molecular force of TSH1 with GCC element is (83.9 ± 2.2) pN. This interaction can be observably blocked by the dissociate TSH1 protein in the solution. These above results proved that TSH1 recognizes and binds to GCC with specifically interaction, which also demonstrate the high sensitivity of the AFM measurements for the single base substitution of the GCC core sequence greatly reduced the binding affinity. Comparing these methods for identification the binding of transcription factor with its DNA element, it was considered that AFM is expected to be a sensitive and reliable method that offers valuable information for the characterization of transcription factors.