Abstract: A method of simultaneous determination of calcium propionate and natamycin in cake by high performance liquid chromatography with programmed wavelength UV detection was developed.The samples were extracted with ultrasound pretreatment,separated with Agilent Zorbax SB- C18( 4.6mm × 150mm) using methanol-di- ammonium hydrogen phosphate( 0.01 mol / m L) as mobile phase with a gradient elution program,determined with variable wavelength detector,and quantified with external standard curve. This method could achieve the separation of calcium propionate and natamycin in cake,and the retention time of calcium propionate and natamycin was( 3.5 ± 0.2) min and( 9.4 ± 0.2) min,respectively.The correlation coefficients of calibration curves were all above 0.9999,and average recoveries were 89% ~ 98%.It could conclude that the method that using HPLC to simultaneously determine calcium propionate and natamycin in cake had characters of quickness,accuracy and high- sensitivity,which could meet the requirements of simultaneously determining calcium propionate and natamycin in cake.