The luminol/sulfobutylether-β-cyclodextrin (SBE(7M)-β-CD) chemiluminescence (CL) system and the interaction of SBE(7M)-β-CD/rutin were first described by flow injection (FI) CL method. It was found that SBE(7M)-β-CD with luminol could form 1:1 complex online, which could accelerate the electrons transferring rate of excited 3-aminophthalate, giving the enhanced CL intensity of luminol. The enhancement of CL intensity was proportional to the concentrations of SBE(7M)- β-CD with a linear range from 25 to 1750 μmol L⁻¹. It was also found that rutin could inhibit the CL intensity from luminol/SBE(7M)-β-CD system, and the decrement of CL intensity was logarithm over the concentrations of rutin ranging from 0.1 to 100.0 nmol L⁻¹, giving the regression equation ΔI = 32.90lgC(rutin) + 16.26 (R² = 0.9952) with a detection limit of 0.03 nmol L⁻¹ (3σ). According to the proposed CL model, the binding constant (K(CD-R)) and the stoichiometric ratio of SBE(7M)-β-CD/rutin complex were obtained as 1.6 × 10⁶ L² mol⁻² and 2:1. The possible mechanism of luminol/SBE(7M)-β- CD/rutin interaction was also discussed. The method was successfully applied to monitor rutin in human urine samples after ingesting SBE(7M)-β-CD/rutin complex, with a total excretion of 68.8% within 8.0 h.