Sediments polluted with high concentrations of persistent organic pollutants, many of which are ligands of the aryl hydrocarbon receptor (AhR), are currently of concern around the industrialized world. Bioassays that can detect the presence of AhR ligands in environmental samples offer a relatively rapid and cost-effective means of prioritizing samples before more elaborate, laborious, and costly chemical analyses are applied. This paper presents a new bioluminescent yeast assay based on transcriptional activation of AhR. Its applicability for determining AhR ligands in complex environmental samples was demonstrated by analyzing a set of sediment samples from the River Kymi, Finland. The results from the assay are shown to be consistent with those from both a chemical analysis and an H4IIE-luc bioassay. The yeast assay procedure is simple and can be performed within 1 day. The yeasts grow rapidly, are easy to handle, and do not require continuous cell culturing. Moreover, the robustness of the yeast allows the application of the test to crude extracts or even sediment suspensions. The yeast assay described in this paper can be useful in screening and prioritization of samples prior to chemical analysis. Moreover, the strain can be used in the construction of fibre-optic biosensors.