Colorimetric sensors are recognized as a promising means for target molecule detection as they provide rapid, cost-effective, and facile sensing visible to the naked eye. Challenges remain though in terms of their detection sensitivity and specificity for short-length target genes. Herein, we demonstrate the successful combination of the catalytic hairpin DNA assembly (CHA) approach with enzyme-linked immunosorbent assay (ELISA)-mimicking techniques for a simple, sensitive, and sequence-specific colorimetric assay to detect short SARS-CoV-2 target cDNA. In the developed CHA-based chemiluminescent assay, a low concentration of target cDNA is continuously recycled to amplify dimeric DNA probes from two biotinylated hairpin DNA until the hairpin DNA is completely consumed. The dimeric DNA probes are effectively immobilized in a neutravidin-coated microplate well and then capture neutravidin-conjugated horseradish peroxidase via biotin-neutravidin interactions, resulting in a sensitive and selective colorless-to-blue color change. The developed sensing system exhibits a high sensitivity with a detection limit of ~1 nM for target cDNA as well as the ability to precisely distinguish a single-base mismatched mutant gene within 2 h. As the proposed system does not require complex protocols or expensive equipment to amplify target cDNA, it has the potential to be utilized as a powerful tool to improve the detection sensitivity of target genes for clinical diagnostics with colorimetric detection.