Protamine is an arginine rich cationic protein and is known to be a very important pharmaceutical compound which is utilized in interventional radiology procedures, vascular surgery, and cardiac surgery, and is the only clinically approved antidote for Heparin overdose. Considering its importance in various biological processes, it is very important to develop sensor platforms for selective and sensitive detection of Protamine. Herein,we present a rarely reported ratiometric fluorescence detection scheme for Protamine, which is based on Protamineinduced disassembly of a dye-polyelectrolyte supramolecular assembly that causes a tuning of the monomeraggregate equilibrium of the dye, and yields a sensitive ratiometric response for the Protamine. Our sensor system registers high sensitivity and selectivity for Protamine. Besides being simple, selective and sensitive, one of the prime features of this developed detection method is its ratiometric response which principally provides increased accuracy for quantitative analysis in complex samples due to inherent internal calibration of two bands. Another noteworthy feature associated with this detection scheme is that it employs commercially available probe molecule and other components that avoid the heavy dependence on time-consuming and tedious synthetic procedures involved with the previously existing methods. Importantly, our current sensing scheme also demonstrates sensing performance in complex human serum matrix.