Competitive host-guest recognition has been utilized to determine small molecules using macrocyclic supra molecular host, while less studies focused on the specific recognition and sensing of protein. In the present work, we are the first time to report a label-free fluorescent assay for insulin determination based on the supramolecular recognition between cationic pillar[6]arene (CP6) and insulin. The approach is based on fluorescence resonance energy transfer (FRET) through competitive recognition between CP6 functionalized reduced graphene oxide (CP6@rGO) and probe/insulin molecules. Probe molecule (RhB) has strong fluorescent signal, and its fluorescent is quenched by rGO based on FRET. When target protein molecule (insulin) is added to CP6@rGO, the probe is displaced by insulin and a host-guest complex CP6@rGO/insulin is formed, resulting in a "turn-on" fluorescence signal. The fluorescence intensity of complex increased linearly with the increase of insulin concentration ranging 0.01-0.50 and 1.0-16.0 mu M, respectively with a detection limit of 3 nM. The sensor was successfully utilized to determine insulin in artificial serum. The molecular docking result showed that the N-terminal Phe of insulin's B chain was included in the CP6 cavity through electrostatic interaction and formed a stable host-guest complex.