DNA hybridization is of tremendous importance in biology, bionanotechnology, and biophysics. Molecular beacons are engineered DNA hairpins with a fluorophore and a quencher labeled on each of the two ends. A target DNA can open the hairpin to give an increased fluorescence signal. To date, the majority of molecular beacon detections have been performed only in aqueous buffers. We describe herein DNA detection in nine different organic solvents, methanol, ethanol, isopropanol, acetonitrile, formamide, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethylene glycol, and glycerol, varying each up to 75% (v/v). In comparison with detection in water, the detection in organic solvents showed several important features. First, the molecular beacon hybridizes to its target DNA in the presence of all nine solvents up to a certain percentage. Second, the rate of this hybridization was significantly faster in most organic solvents compared with water. For example, in 56% ethanol, the beacon showed a 70-fold rate enhancement. Third, the ability of the molecular beacon to discriminate single-base mismatch is still maintained. Lastly, the DNA melting temperature in the organic solvents showed a solvent concentration-dependent decrease. This study suggests that molecular beacons can be used for applications where organic solvents must be involved or organic solvents can be intentionally added to improve the molecular beacon performance.