Abstract: The purpose was to establish a method for determining the content of vitamin B6 in dry powder of corn steep liquor using HPLC.The detection conditions were:using Symmetry-C18 column (3.9mm×150mm, 5μm) , isocratic elution (A/B, 1∶6) and mobile phase A:methanol, phase B:2.5% acetic acid solution containing 0.05g/L EDTA and 1.1g/L sodium heptanesulfonate (pH3.40) , flow rate :0.8mL/min, detection wavelength :280nm, sample analysis time was 10min.The linear ranges of vitamin B6 were 0.10 to 100μg/mL (r=0.9999) , relative standard deviation was less than 15%, recovery of the three concentration of vitamin B6 were 93.66%±1.38%, 95.60%±2.77%, 93.52%±2.46% (n=4) .The method was simple, rapid and reliable.It could be used in determination of vitamin B6 in dry powder of corn steep liquor.