An analytical method based on dispersive solid phase extraction/liquid chromatography-tandem mass spectrometry（dSPE/LC-MS/MS） was developed for the determination of amitraz,semiamitraz（DMPF） and their metabolites N-（2,4-dimethylphenyl） formamide（DMF） and 2,4 dimethylaniline（DMA） residues in royal jelly.The royal jelly samples were diluted with a buffer solution（pH 9.0） ,and the protein in royal jelly was precipitated with acetonitrile and NaCl.The acetonitrile supernatant was cleaned up with primary secondary amine（PSA） ,C18 and anhydrous MgSO4 by dSPE method,then the supernatant solution was filtered through a nylon membrane（0.22 μm） into a glass LC vial for LC-MS/MS analysis.The solution was separated on an Agilent Eclipse XDB-C18 column（150 mm×4.6 mm,5 μm） with acetonitrile-0.15%formic acid solution as mobile phases by gradient elution,then determined by LC-MS/MS with electrospray ionization（ESI） in positive ion scanning mode under multiple reaction monitoring（MRM） mode,and finally quantified by isotope internal standard method.Results showed that there were good linear relationships for amitraz,DMPF,DMF and DMA in the concentration range of 0-100 μg/kg with their correlation coefficients（r） more than 0.996.The limits of quantitation（S/N=10） for the analytes were 0.10,0.25,5.0 and 2.5 μg/kg,respectively.The spiked recoveries at three levels of 5,10 and 20 μg/kg were in the range of 77.0%-107%with relative standard deviations（RSD） of 2.0%-11%.This method was rapid and convenient,and was suitable for the simultaneous determination of amitraz,DMPF and their metabolites residues in royal jelly samples.