Recent techniques for quantification of dioxins and dioxin-like compounds involve mainly the costly and time-consuming high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). To ensure environmental safety, it is extremely crucial to develop high-throughput assays for screening potential contamination of dioxin-like chemicals in samples from various matrices. The Chemical Activated LUciferase eXpression (CALUX) bioassay is an in vitro luciferase-reporter-gene assay for detecting the Toxic Equivalents (TEQ) levels of dioxins and dioxin-like compounds based on their high affinity with aryl hydrocarbon receptor (AhR). As compared with HRGC/HRMS, CALUX bioassay is a fast and low-cost method for high-throughput screening of those highly dioxin-contaminated samples from environment and foods. We have established a recombinant adenovirus carrying a DRE-driven luciferase gene (Ad-DRE-Luc) and use a rat hepatoma cell line H4IIE as the host. On the basis of this DRE-driven luciferase assay system, this project aims to develop a high-throughput screening bioassay for PCDD/Fs measurement in samples from various matrices. In this study, we have established a fast sample cleanup system and the standard cell culture procedure for the assay. After fitted into the Hill equation, the regression curve of 2,3,7,8-TCDD was shown with R2 ≥ 0.995. The DRE-driven luciferase assay system was tested with liquid standards (EDF-5416 and EDF-5417), a certified reference material (CRM) (DX-1), samples from soil and sediment, and a variety of bio-samples. Our results showed that the sensitivity of Ad-DRE-Luc/H4IIE system in dioxin measurement is significantly higher than that of the previous Huh7-DRE-Luc system. The Ad-DRE-Luc/H4IIE system has been demonstrated to be a powerful technique for pre-screening dioxin samples from different sources, including organisms.