Ragi bifunctional α-amylase-trypsin inhibitor (RBI) of Eleusine coracana (L.) Gaertn. (finger millet) simultaneously inhibits α-amylase and trypsin. In continuation of previous work on the cloning, expression and characterization of RBI, a bidirectional promoter from finger millet was explored on the basis of experimental observations. Two trypsin inhibitors were identified while purifying RBI from a trypsin-Sepharose column eluent. Using an FPLC gel filtration column, these three inhibitors were purified to homogeneity and subjected to MALDI-TOF-TOF-MS/MS analysis and N-terminal sequencing. Both ragi trypsin inhibitors (RTIs) showed the same N-terminal sequence and considerable sequence similarity to RBI, indicating the presence of a multigene protease inhibitor family in finger millet. To gain insight into the evolution of these genes, the upstream region of RBI was explored by Genome Walking. Interestingly, on sequencing, a genome walking product of ∼1 Kb showed presence of an N-terminal RBI specific primer sequence twice but in opposite directions and leaving an intervening region of ∼0.9 Kb. The intervening region was presumed to represent an E. coracana bidirectional promoter (EcBDP), intuitively having a divergent RBI-RTI gene pair at two sides. For assaying the bidirectionality of promoter activity, a dual reporter GUS-GFP vector construct was made for plant expression containing the reporter genes at two ends of EcBDP, which was used to transform Agrobacterium tumefaciens LBA 4404. Transient plant transformation by recombinant Agrobacterium cells was carried out in onion scale epidermal cells and finger millet seedling leaves. Simultaneous expression of GUS and GFP under EcBDP established it as a potent natural bidirectional promoter from monocot origin, thereby potentially having vast application in cereal gene manipulations. In addition, inducibility of the EcBDP by either abscisic acid or cold treatment, as determined by transient transformation in onion, would substantiate more precise control of gene expression to mitigate the effects of adverse environmental conditions.