The method of calculating theoretical content of triacylglycerols with ECN42 in olive oil was introduced, which distinguished adulteration of olive oil by determination of the absolute difference between actual and theoretical content of triacylglycerols with ECN42（△ECN42）.The HPLC condition to quantify ECN42, and the reproducibility of the method was researched. The results showed that using differential refractometer as the detector, column temperature 38℃, mobile phase acetone/acetonitrile（50∶50，V/V），flow rate 1.5 mL/min, under the condition, it showed a good chromatographic separation effect with good repeatability, and RSD less than 1% and SR between 0.02 and 0.11. It provided basis for instituting national standard.