Supramolecular host-guest interaction and sensing between cationic pillar[5]arenes (CP5) and L-carnitine were developed by the competitive host-guest recognition for the first time. The fluorescence sensing platform was constructed by CP5 functionalized Au nanoparticles (CP5@Au-NPs) as receptor and probe (rhodamine 123, R123), which shown high sensitivity and selectivity for L-carnitine detection. Due to the negative charge and molecular size properties of L-carnitine, it can be highly captured by the CP5 via electrostatic interactions and hydrophobic interactions. The host-guest mechanism between PP5 and L-carnitine was studied by 1H NMR and molecular docking, indicating that more affinity binding force of CP5 with L-carnitine. Therefore, a selective and sensitive fluorescent method was developed. It has a linear response of 0.1–2.0 and 2.0–25.0 μM and a detection limit of 0.067 μM (S/N = 3). The fluorescent sensing platform was also used to detect L-carnitine in human serum and milk samples, which provided potential applications for the detection of drugs abuse and had path for guarding a serious food safety issues.