A high performance liquid chromatographic(HPLC) method was established for the simultaneous determination of trans,trans-muconic acid(tt-MA)，hippuric acid(HA)，mandelic acid(MA)，phenylglyoxy acid(PGA)，p-aminophenol(PAP)and p-nitrophenol(PNP)in urine．The chromatographic separation and the detection conditions were optimized，and a programmed gradient which the wavelength was changed with the time was applied for the detection．The optimal pretreatment conditions for the sample were as follows:extraction solvent: 5 mL dichloromethane-isopropanol(7∶3)，sample volume:5 mL，NaCl:0.75 g，extraction time:2 min．The sample was firstly extracted under acidic condition，and repeated once under the neutral condition，then the two extraction liquids were merged．The extract liquor was volatilized to dryness in water bath with the volatilization temperature less than 70 ℃，then redissolved with 1.0 mL mobile phase.Under the optimal conditions， the linear ranges of the six components were up to 103，and the correlation coefficients(r) were not less than 0.999 3．The detection limits(S/N=3) were in the range of 3-30 μg/L．The spiked recoveries were in the range of 86%- 104% with relative standard deviations less than 5%．The method was sensitive，accurate and rapid，and could meet the needs of biological monitoring on the metabolites of harmful substances in urine.