A high performance liquid chromatographic(HPLC)method combined with on-line clean-up and solid-phase extraction(SPE) was developed for the fast and effective determination of trace amount of vitamin B12(VB12) in beverages.A small change in the flow scheme of the traditional on-line SPE system reverses the flush direction on the SPE column，and creates an online SPE system with dual function(trapping the analytes and partially separating it from interferences prior to the analytical column) that eliminates interferences much more efficiently.The trapping of VB12 was completed by injecting 2.5 mL beverage sample into the SPE column(Thermo Scientific Acclaim PA Ⅱ，3 μm，3.0 mm×33 mm)，using phosphate buffer-acetonitrile as mobile phase,and then the VB12 was partially separated on the SPE column by changing mobile phase gradient.Therefore,only those interferences co-eluting with VB12 entered the analytical column，and more interferences were removed.The volume of VB12 cut from the SPE column was separated on the analytical column(Thermo Scientific Acclaim PA Ⅱ，3 μm，3.0 mm×150 mm) using phosphate buffer-acetonitrile as mobile phase，and detected with UV detector at 361 nm.The complete analysis only took 20 min.The relative standard deviation(RSD) of VB12 was 0.027% for retention time,and 2.2% for peak area.Good linearity was obtained in the range of 0.1-5.0 μg/L with correlation coefficient of 0.999 9.The spiked recoveries for 4 beverage samples were in the range of 91%-109%,and the limit of detection for this method was 0.046 μg/L.Compared with the direct large-volume injection and the traditional on-line SPE system,this method was better expression in easy manipulation and effective clean-up.