BACKGROUND: Semi-specific microbial biochemical oxygen demand (BOD) biosensors were constructed using living cells of Escherichia coil R17.1.3 (E. coil) and Raoultella terrigena P74.3 (R. terrigena) immobilized in agarose gel matrix. The research involved comparison with the Pseudomonas fluorescens P75 (P. fluorescens) biosensor that had no specificity in decomposing lactose and milk derivates. The constructed BOD biosensors were calibrated with OECD synthetic wastewater and tested with different wastewater samples. RESULTS: The linear range of the calibration curve was 5-200 mg L-1 BOD7 for R. terrigena and P. fluorescens based biosensors and 150 mg L-1 BOD7 for the E. coil based biosensor. Service life was 60 days for E. coil and P. fluorescens based biosensors and 40 days for R. terrigena based biosensors. BOD values for dairy industry wastewater obtained with current semi-specific biosensors considerably overestimate BOD7, while universal biosensors underestimate BOD7 obtained by the conventional 7-day BOD test. CONCLUSION: In spite of extensive overestimation of BOD7 the semi-specific biosensors enabled better estimation of BOD in dairy industry wastewater than a reference P. fluorescens biosensor. The best result, in terms of service life, stability, sensitivity and reproducibility was accomplished with semi-specific E.coli biosensor. (C) 2010 Society of Chemical Industry