In computational molecular biology, gene regulatory binding sites prediction in whole genome remains a challenge for the researchers. Now a days, the genome wide regulatory binding site prediction tools required either direct pattern sequence or weight matrix. Although there are known transcription factor binding sites databases available for genome wide prediction but no tool is available which can construct different weight matrices as per need of user or tools available for large data set scanning by first aligning the input upstream or promoter sequences and than construct the matrices in different level and file format. Considering this, we developed a DNA MATRIX tool for searching putative regulatory binding sites in gene upstream sequences. This tool uses the simple biological rule based heuristic algorithm for weight matrix construction, which can be transformed into different formats after motif alignment and therefore provides the possibility to identify the most potential conserved binding sites in the regulated genes. The user may construct and save specific weight or frequency matrices in different form and file formats based on user based selection of conserved aligned block of short sequences ranges from 6 to 20 base pairs and prior nucleotide frequency before weight scoring.