Hybridization probes have been used for the detection of single nucleotide variations (SNV) in DNA and RNA sequences in the mix-and-read formats. Among the most conventional are Taqman probes, which require expensive quantitative polymerase chain reaction (qPCR) instruments with melting capabilities. More affordable isothermal amplification format requires hybridization probes that can selectively detect SNVs isothermally. Here we designed a split DNA aptamer (SDA) hybridization probe based on a recently reported DNA sequence that binds a dapoxyl dye and increases its fluorescence ( Kato, T.; Shimada, I.; Kimura, R.; Hyuga, M., Light-up fluorophore-DNA aptamer pair for label-free turn-on aptamer sensors. Chem. Commun. 2016 , 52 , 4041 - 4044 ). SDA uses two DNA strands that have low affinity to the dapoxyl dye unless hybridized to abutting positions at a specific analyte and form a dye-binding site, which is accompanied by up to a 120-fold increase in fluorescence. SDA differentiates SNV in the inhA gene of Mycobacterium tuberculosis at ambient temperatures and detects a conserved region of the Zika virus after isothermal nucleic acid sequence based amplification (NASBA) reaction. The approach reported here can be used for detection of isothermal amplification products in the mix-and-read format as an alternative to qPCR.