A highly sensitive sensing system for heparin has been developed using a synthetic fluorescent dye, L-phenylalanyl-L-phenylalanine-modified dansyl (FF-Dns), in combination with the cationic surfactant octadecyltrimethylammonium chloride (OTAC). At low concentrations, FF-Dns and OTAC self-assemble, leading to a reduction in the fluorescence intensity of the dansyl group. However, upon the introduction of heparin, the electrostatic binding between OTAC and heparin disrupts the self-assembly, effectively releasing FF-Dns and restoring its fluorescence. This system shows exceptional selectivity for heparin over a range of potential interfering substances. The detection limit for heparin was determined to be 31.7 ng mL^-1, with a linear response range of 0-4 μg mL^-1. In addition, a "turn-on" fluorescence-based assay platform using FF-Dns/OTAC was successfully established in 0.2% serum with a detection limit of 30.57 ng mL^-1 and a linear span of 0-5 μg mL^-1. In addition, the FF-Dns/OTAC/heparin mixture was identified as a potential sensing platform for protamine, exploiting the strong binding affinity between protamine and heparin.