In this work, the constructed bioluminescent Acinetobacter strain DF4/PUTK2 was employed to assess the toxicity of phenolic compounds and the 5 min EC50 values were calculated. The results of the DF4/PUTK2 assay were further evaluated by comparing with the results of the Vibrio fischeri luminescence inhibition assay. To develop a bioassay system appropriate to be used in continuous toxicity testing, strain DF4/PUTK2 was subjected for immobilization in microtiter plates into the matrices Ca-alginate, polyacrylamide, agar and agarose. After a choice of materials was tried, Ca-alginate was selected as the most suitable candidate material. Because, it could be stored at least 8 weeks at 4 degrees C, during which the ability of the bioreporter DF4/PUTK2 to detect the toxicity of phenolics was maintained. However, the stability of the bioluminescence for DF4/PUTK2 cells immobilized into agarose and agar was significantly less than that of cells stored in alginate suspensions. This study recommended that luxCDABE-marked Acinetobacter strain DF4/PUTK2 could be employed to assay the ecotoxicity of environmental samples contaminated with phenols. The host strain of the bioreporter DF4/PUTK2 is Acinetobacter strain DF4. It is known that members of the genus Acinetobacter are widespread in nature and also involved in biodegradation, leaching and removal of several organic and inorganic man-made hazardous wastes.