To identify and apply tissue-specific promoters is one of the major challenges in plants genetic engineering for optimizing efficient expression of interest genes in appropriate tissues. In this research, open-source database information of Arabidopsis thaliana was adapted to determine root-specific expressed promoter region. A total seven sequences that might function as a root-specific promoter element were initially isolated from Arabidopsis genomic DNA. Then seven promoters were cloned into pBGWFS7 in which β-glucuronidase (gus) and green fluorescent protein (gfp) genes were linked. The GUS activities were measured in different tissues of transgenic Arabidopsis by both histochemical GUS staining and fluorescent 4 methylumbelliferyl β-D-glucuronide (MUG) assay. To confirm root-specific expression, GFP-confocal microscope analysis was conducted in Arabidopsis transgenic plant. As a result, the five promoters showed strong GUS activity in the root tissue as compared with the CaMV35S promoter. To test crop application availability as a root-specific promoter, seven promoters were introduced into tomato plants and confirmed transient expression using Agrobacterium rhizogenesis ARqua1 root-nodule inducible strain. Two promoters showed that gus genes were specifically expressed in roots of transgenic tomato plant. Taken together, the novel seven promoters showed specific activity in root suggesting that it is applicable in crop improvement.