The photophysical behavior of luminol (LH(2)) was studied in a variety of biologically relevant systems ranging from surfactants, cyclodextrin, and proteins using steady-state and time-resolved fluorescence spectroscopy. It was shown that, out of two possible LH(2) conformers present in solution, the sequestration of relatively less polar structure into the hydrophobic domain of biological media is the primary reason for decrease in fluorescence intensity. The efficacy of LH(2) fluorescence quenching is substantially higher in micellar subdomain of cationic surfactant and depends on the nature of the headgroup. The thermodynamic parameters like enthalpy (Delta H) and entropy (Delta S) change, etc., corresponding to the binding of LH(2) in the model water-soluble protein, bovine serum albumin (BSA), were estimated by performing the fluorescence titration experiment at different temperatures. The involvement of subdomain IA and IIA of BSA in LH(2) binding was confirmed from the ligand replacement process with bilirubin (BIL). The difference in ligand binding with structurally homologous human serum albumin (HSA) is discussed in terms of positive cooperativity among these two binding domains of BSA with a Hill coefficient (n(H)) value of 2.26 +/- 0.18 and a half-maximal concentration (K(0.5)) of 5.74 +/- 0.23 mu M at 298 K.