Dyeing effluent is a typical refractory wastewater containing toxic pollutants. It is difficult to treat it to meet discharge regulations. The biotoxic effects of pollutants on microorganisms are one of the main constraints on efficient wastewater degradation. The aim of this study was to evaluate biotoxic effects and try to screen toxic substances from dye wastewater. The toxic effects of wastewaters collected at different treatment stages from a wastewater treatment plant in a dye industrial park were determined using bioluminescent bacteria (Vibrio qinghaiensis sp.-Q67). Toxic substances from both influent and effluents were identified by gas chromatography-mass spectrometry. Spearman's rank correlation coefficients indicated significant positive correlations between the toxicity values, and chemical oxygen demand (COD), NH3-N, and total nitrogen (TN). After identifying the toxic levels, phenol, pyridine, and esters were confirmed as the principal organic pollutants in dye wastewater, and increase its toxicity.