Display omitted •Anisotropy study reveals incorporation of OFX inside the cavity of β-CD.•Negative value of ΔH and positive value of ΔS validate that hydrophobic interaction in complex.•Negative value of ΔG suggest the complex is spontaneous in nature. Ofloxacin (OFX) which is poorly soluble fluroquinolone drug exists as cationic form in acidic pH and zwitterionic form in neutral pH. The behaviour of host-guest interaction of Ofloxacin (OFX) with β-Cyclodextrin (β-CD) has been investigated by fluorescence quenching method in acidic and neutral medium using copper as quencher at various temperatures. Using the Benesi−Hildebrand relationship, the stochiometry of the host-guest complexation has been estimated. The incorporation of OFX inside the cavity of β-CD was revealed from fluorescence anisotropy measurement. Effect of temperature on the Stern-Volmer quenching constant (KSV) and binding constant (K) has been analyzed and a suitable mechanism has been proposed on the nature of quenching. Using Vant-Hoff equation and Gibbs-Helmholtz equation, thermodynamic parameter change of enthalpy (ΔH), entropy (ΔS) and Free Energy (ΔG) has been determined, negative value of ΔH and positive value of ΔS validate that hydrophobic interaction between drug and quencher and negative value of ΔG suggest the complex is spontaneous in nature.