In the present study, the interaction of two structurally related proteolytic enzymes trypsin and alpha-chymotrypsin (CHT) with 4',6-Diamidino-2-phenylindole (DAPI) has been addressed. The binding of DAPI to CHT has been characterized by steady-state and picosecond time-resolved spectroscopic techniques. Enzymatic activity of CHT and simultaneous binding of the well-known inhibitor proflavin (PF) in the presence of DAPI clearly rule out the possibility of DAPI binding at the catalytic site of the enzyme. The spectral overlap between the emission of DAPI and absorption of PF offers the opportunity to explore the binding site of DAPI using Förster resonance energy transfer (FRET). FRET studies between DAPI and PF indicate that DAPI is bound to CHT with its transition dipole nearly perpendicular to that of PF. Competitive binding of DAPI with another fluorescent probe 2,6-p-toluidinonaphthalene sulfonate (TNS), having a well-defined binding site, indicates that DAPI and TNS bind at the same hydrophobic site of the enzyme CHT. The difference in the interactions of two well-studied, structurally similar enzymes with the same molecule may find its application in the design of specific substrate mimics or inhibitors of the enzymes.