Fast, sensitive, specific, and user-friendly DNA assay is a key technique for the next generation point-of-care molecular diagnosis. However, high-cost, time-consuming, and complicated enzyme-based DNA amplification step is essential to achieve high sensitivity. Herein, a short target DNA-catalyzed formation of quantum dot (QD)DNA hydrogel is proposed as a new DNA assay platform satisfying the above requirements. A single-stranded target DNA catalyzes the opening cycle of DNA hairpin loops, which are quickly self-assembled with DNAfunctionalized QDs to generate QD-DNA hydrogel. The three-dimensional hydrogel network allows efficient resonance energy transfer, dramatically lowering the limit of detection down to similar to 6 fM without enzymatic DNA amplification. The QD-DNA hydrogel also enables a rapid detection (1 h) with high specificity even for a singlebase mismatch. The clinical applicability of the QD-DNA hydrogel is demonstrated for the Klebsiella pneumoniae carbapenemase gene, one of the key targets of drug-resistant pathogenic bacteria.