The invention relates to a method for rapidly separating and determining water-soluble B-vitamins in infant food and dairy products. The method comprises the following steps: preparing a standard working solvent: weighing an appropriate amount of vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B9 and vitamin B12 standard substances, placing into a brown volumetric flask, reaching a constant volume of 100mL by virtue of ultra-pure water, and freezing storing; preparing a sample working solvent: weighing 3 to 8 g of samples, adding 50 to 70 mL of 0.1mol/L hydrochloric acid, uniformly mixing, sealing, placing into a high-pressure sterilizing pot, keeping for 25 to 35 min at 120 to 125 DEG C, dissolving, and performing pretreatment for the sample by adopting an enzymolysis method; and analyzing by adopting a liquid-phase chromatograph-mass spectrometry. The method can analyze and detect seven water-soluble B-vitamins, ensure the recovery rate of each component and save the detection time and cost.