An ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method was established to determine N-（1，3-dimethylbutyl）-N′-phenyl-p-phenylenediamine-quinone （6PPD-Q） in human urine and dust in order to understand the internal and external exposure levels in humans. The sample preparation conditions were systematically investigated and the chromatographic conditions and MS parameters were optimized. Briefly， internal standard ¹³C₆-6PPD-Q （0.1 ng） was added to a urine sample. Glutathione and NaCl were added after equilibration for 30 min. The mixture was then twice ultrasonically extracted with ethyl acetate. Internal standard ¹³C₆-6PPD-Q （1.0 ng） was also added to a dust sample and the mixture was twice ultrasonically extracted with n-hexane. The combined organic phases were concentrated to near-dryness and then redissolved for instrumental determination， which was performed on a Phenomenex Kinetex F5 column （100 mm×3 mm， 2.6 μm）， with the target analyte gradient eluted using 10 mmol/L ammonium acetate aqueous solution containing 0.01% formic acid and acetonitrile. Positive electrospray ionization （ESI⁺） and multiple reaction monitoring （MRM） modes were used for identification purposes， and an isotope-labeled internal standard was added for quantification. Good linearities were achieved under the optimized conditions within the 0.01–4.00 and 0.01–20.0 μg/L ranges for urine and dust， respectively， with correlation coefficients of 0.999 9 and 0.999 3， respectively. Limits of detection （LODs） were 0.6 ng/L （urine） and 0.018 ng/g （dust）. Spiked recoveries of 6PPD-Q were 90.3%–94.1% at low， medium， and high spiked levels， with intra-day and inter-day precisions of 0.9%–5.9% and 1.1%–6.3%， respectively. Matrix-effect investigations revealed that 6PPD-Q exhibited weak matrix effects in urine and dust after correction with the isotopic internal standard. The developed method was used to analyze 120 human urine samples， which led to a 6PPD-Q detection frequency of 74.2%， with mass concentrations ranging from <LOD to 13 ng/L， with average and median mass concentrations of 2 and 1 ng/L， respectively. However， 6PPD-Q was detected with a frequency of 100% in 31 indoor dust samples with contents of 1.8–24.9 ng/g， and average and median contents of 5.23 –3.05 ng/g， respectively. The developed method is accurate， reliable， highly sensitive， and it is suitable for the rapid determination of 6PPD-Q in human urine and dust samples.