Abstract: Objective To construct the PXR response element luciferase reporter. Methods The pentamer-sequences of (Everted repeat elelment-6, ER-6 element)₅and (Direct repeat element-3, DR-3 element)₅were gained from chemical synthesis method. The pentamer-sequences of PXR response elements (ER-6 or DR-3) were cloned into pGL3-Promoter vectors. The transcriptional activity of PXR was measured by luciferase assay. Results The PXR response elements luciferase reporter was successfully constructed. The Anisomycin, which was the agonist of PXR, could induce the activity of ER6-Luc (R²=0.95; P=0.002 2) and DR3-Luc (R²=0.96; P=0.000 91) reporters in a dose dependent manner with the EC₅₀value of (0.11±0.04)μmol/L and (0.13±0.06)μmol/L, respectively. The Ketoconazole, which was the antagonist of PXR, could inhibit the activity of ER6-Luc (R²=0.97; P=0.000 85) and DR3-Luc (R²=0.98; P=0.000 11) reporters induced by Anisomycin in a dose dependent manner with the EC₅₀value of (0.71±0.11)μmol/L and (1.73±0.15)μmol/L, respectively. Conclusion The PXR response elements luciferase reporter is successfully constructed and the way to detect the transcriptional activity of PXR is established.