The design and feasibility of split G-quadruplex-based DNA nanotweezers (split Gq-based DNA-NTs) that can recognize short nucleic acids with a single-base mismatch are discussed. The split Gq-based DNA-NTs consist of three single-stranded DNA sequences forming a tweezers shape with split Gq sequences at the edge of each arm. In response to target recognition such as specific nucleic acids, the split Gq sequences come close to each other and then regain their ability to form a Gq/hemin complex, which exhibits peroxidase activity. Therefore, specific nucleic acids can be detected by measuring the peroxidase activity of split Gq-based DNA-NTs in a homogeneous assay format. Although split Gq-based DNA-NTs have been utilized as a biosensing molecule that recognizes a specific target and generates a sensing signal, the condition of target recognition was not well elucidated. Therefore, it was yet unclear whether the split Gq-based DNA-NTs can recognize short nucleic acids with a single-base mismatch. In this study, by selecting microRNA (miRNA) sequences of let-7a and let-7c as a single-base mismatch model, we successfully developed split Gq-based DNA–bridged nucleic acid chimera NTs (split Gq-based DNA–BNA chimera NTs) that can distinguish these miRNAs on the basis of the difference in melting temperature. It is expected that the split Gq-based DNA–BNA chimera NTs will be applicable to the development of point-of-care testing devices as target recognition and signal generation elements.