Many molecular biology experiments and clinical diagnostics rely on the detection or confirmation of specific nucleic acid sequences. Most DNA or RNA detection assays utilize radioactive or fluorescence labeling but although these tags are sensitive, safety issues (in the case of radiolabeling) or the need for expensive instrumentation (such as a fluorimeter or radiometric detector and the associated image analyzer softwares) can sometimes become impediments for some laboratories to use these detection tags. G-quadruplexes have emerged as efficient DNA-based peroxidases that can convert colorless compounds, such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), into colored products and therefore are excellent platforms to use to detect nucleic acids via colorimetric means. Here, we describe the detection of a single-stranded DNA template using a split G-quadruplex probe that is catalytically non-proficient but becomes active after being reconstituted upon binding to the target template.