The development of a new technology for the detection of doublestranded (ds) DNA enables multiple biomedical applications including identifying multiple pathogens simultaneously. We previously employed colorimetric SEquence-Enabled Reassembly with TEM-1 β-lacatamase (SEER-LAC) to detect specific bacterial DNA sequence. SEER-Lac consists of the two inactive β-lactamase fragments which of each attached to a zinc finger protein (ZFP) would reassemble into an active full-length enzyme upon ZFPs binding to its target DNA. Here, we engineered two pairs of ZFPs which of each recognizes shiga toxin in E. coli O157 and staphylococcal enterotoxin B in Staphylococus Aureus, respectively. Biotin was simply conjugated to the detection probe ZFP, which allows for generating chemiluminescent signal in streptavidin-HRP (Horseradish peroxidase) assay upon ZFPs binding to their target DNA. Our assay generates DNA-dependent signal and allows for a detection limit of 0.5 nM without DNA amplification or DNA labeling. Our system can be developed into a simple multiplexed detection diagnostic for multiplexed detection of dsDNA.