Fluorescence of quinolones including norfloxacin, ciprofloxacin and S- and R-ofloxacin is quenched upon association with single and double-stranded DNA (ss- and ds-DNA). The ratios of fluorescence intensity in the presence of DNA to its absent were plotted with respect to the DNA concentration to construct the Stern-Volmer plot. The slope of the Stern-Volmer plot become larger as the temperature is lowered, ensuring that the fluorescence quenching is static process, i.e., the fluorescence is quenched by formation of the non-fluorescent complex between quinolone and DNA. In the static quenching mechanism, the quenching constant which is equivalent to the slope of the Stern-Volmer plot, is considered as the equilibrium constant for the association of quinolones and DNA. From the temperature-dependent equilibrium constant, ${Delta}H^0;and;{Delta}S^0$ was obtained using the van’t Hoff relation. In general, association of the quinolone with ds- as well as ss-DNA is energetically favorable (an exothermic) process while the entropy change was unfavorable. Due to the steric effect of the substituents, the effect of the quinolone ring is smaller on the ss-DNA compared to ds-DNA.