Herein, we have studied the analysis of fluorescence quenching for newly synthesized biologically active 3(2H)-pyridazinone derivative 5-(5-bromo-2-hydroxy-phenyl)-2-phenyl-2H-pyridazin-3-one [BHP] by various concentrations of aniline using UV-Visible spectroscopy, fluorescence spectroscopy and time-correlated single photon counting technique in five different solvents namely, methanol, ethanol, propan-2-ol, dimethylsulfoxide and ethyl acetate at room temperature. The fluorescence intensity of BHP molecule decrease with increasing in the aniline concentration and it is studied using the Stern-Volmer relation. The obtained Stern-Volmer plots were found to be linear in all the five solvents. The various parameters responsible for the fluorescence quenching such as quenching rate parameters (k ), diffusion rate parameter (k ) and the probability of quenching per encounter (p) were experimentally calculated in all five solvents. An activation energy of quenching (E ) was calculated using the values of activation energy of diffusion (E ) and p. It was found that the values of E are greater than E in all five solvents studied. Further, it is inferred that the fluorescence quenching reactions in BHP molecule are more significantly affected by activation energy processes.