A 5′ flaking nucleotide sequence of 1,101 bp of rice catalase-B gene (CatB) coding sequence having coordinates from −808 to +294 with respect to the transcription start site was transcriptionally fused to the β-glucuronidase gene (GUS), and functionally analyzed through transient and stable expression assay systems to identify the minimal promoter sequence containing the important cis-regulatory regions controlling its expression. The progressive up-stream and down-stream (5′/3′) deletion analysis of the 1,101-bp fragment revealed that minimal nucleotide sequences spanning from −121 to +56 possessed maximum GUS activity, which was nearly six- to eightfold higher than GUS activity driven by CaMV35S promoter. DNase I footprint and gel retardation analysis of the minimal promoter sequence with nuclear extract revealed the presence of a “AAAAG” nucleotide signature referring to DNA-binding with one finger (Dof) transcription factor. In addition, the presence of CAAT-box sequence was also evident. The Dof interacting sequence in CatB promoter of rice has been established for the first time in this work.