We report a facile yet effective strategy for the fluorescence assay of heparin based on assembly and disassembly of the glutathione-protected gold nanoclusters (GSH-Au NCs) modulated via the competitive interaction of cetyltrimethyl ammonium bromide (CTAB) with heparin and GSH. Firstly, CTAB can increase the fluorescence of Au NCs by forming a nanocomposite via electrostatic and hydrophobic self-assembly. By adding heparin to the assay, CTAB is removed from the nanocomposites because of its higher affinity for heparin, quenching the fluorescence signal. Such a fluorescence feature induced via assembly and disassembly of the NCs enables our assay to possess high selectivity and sensitivity. The linear response toward heparin was obtained over the range 0.1–1.6 μg mL⁻¹ with a low detection limit of 0.075 μg mL⁻¹. Furthermore, satisfactory sensing perfomance for heparin in human serum makes this method hold great promise in heparin-related biomedical applications.