In this paper a label-free fluorescent sensor for probing the interaction between heparin and protein was reported. Heparin, the bioactive polyanions, formed supramolecular assemblies with cationic surfactant cetyltrimethyl ammonium bromide (CTAB). The environment-dependent dye pyrene, encapsulated in hydrophobic interiors of the supramolecular assemblies worked as the fluorescence probe. Once the heparin-binding protein was added, competing interactions of protein with heparin would weaken the interaction between CTAB and heparin. As a result, the noncovalently sequestered pyrene would be released upon disassembly and the fluorescence of the released pyrene was subsequently decreased. The binding events were exemplified by protamine and Tat peptide, these processes were also verified by DLS and TEM. Such a strategy is appealing as organic synthesis was traded off against supramolecular assembly. This label-free fluorescent system is simple, selective, convenient, and can serve as a good complement to other existing methods, also this method has the potential for preprimary drug screening.