A method for rapid determination of Vitamin B 12 in functional drinks by online SPE coupled with dual gradient high performance liquid chromatography and dual valves was developed. First,an aliquot of 2. 5 mL functional drink sample was injected directly into the SPE column with loading pump,and Vitamin B 12 was concentrated in the SPE column,while most interfering material was removed; After cleanup,the valve was switched and retained Vitamin B 12 was flushed to analytical column. The online SPE column was Thermofisher IonPac NG1( 4 mm × 35 mm), the analysis column was Thermofisher Acclaim PAII C18( 4. 6 × 150 mm,5 μm),two columns were used at 30℃. The mobile phase was a gradient mixer of acetonitrile and 0. 025% TFA( volume to volume),and the detection wavelength was set at 220 nm. The injection volumes were 1 000 μL for each run. Based on the above optimizing chromatogram condition,Vitamin B 12 was separated satisfactorily. The limitation of detection could be 0. 54 μg / L,the recovery could be 101. 29%.