In this study, an automated sequential injection lab-on-valve (SI-LOV) system was designed for the on-line matrix removal and preconcentration of quercetin. Octadecyl functionalized magnetic silica nanoparticles were prepared and packed into the microcolumn of the LOV as adsorbents. After being adsorbed through hydrophobic interaction, the analyte was eluted and subsequently introduced into the electrochemical flow cell by voltammetric quantification. The main parameters affecting the performance of solid-phase extraction, such as sample pH and flow rate, eluent solution and volume, accumulation potential and accumulation time were investigated in detail. Under the optimum experimental conditions, a linear calibration curve was obtained in the range of 1.0 × 10⁻⁸ to 1 × 10⁻⁵ mol L⁻¹ with R² = 0.9979. The limit of detection (LOD) and limit of quantitation (LOQ) were 1.3 × 10⁻⁹ and 4.3 × 10⁻⁹ mol L⁻¹, respectively. The relative standard deviation (RSD) for the determination of 1.0 × 10⁻⁶ mol L⁻¹ quercetin was found to be 2.9% (n = 11) along with a sampling frequency of 40 h⁻¹. The applicability and reliability of the automated method described here had been applied to the determination of quercetin in human urine and red wine samples through recovery experiments, and the obtained results were in good agreement with those obtained by the HPLC method.