Quantification of extractable antibiotics in soils is important to assessing their bioavailability and mobility, and ultimately their ecotoxicological and health risks. This study aimed to establish a biosensor method for detecting extractable tetracyclines in soils (Alfisol, Mollisol, and Ultisol) using whole-cell biosensors containing a reporter plasmid (pMTGFP or pMTmCherry) carrying fluorescent protein genes tightly controlled by tetracyclines-responsive control region (tetRO). This whole-cell biosensor method can simultaneously measure 96 or more samples within 6 h and is easily parallelizable, whereas a typical high-performance liquid chromatography (HPLC) method may require 7 times more of analysis time and much greater cost to achieve similar analytical throughput. The biosensor method had a detection limit for each of six tetracyclines between 5.32-10.2 μg/kg soil, which is considered adequate for detecting tetracyclines in ethylenediaminetetraacetic acid (EDTA) extracts of soils. Relative standard deviation was between 19.8-51.2% for the biosensor Escherichia coli DH5α/pMTGFP and 2.98-25.8% for E. coli DH5α/pMTmCherry, respectively, suggesting that E. coli DH5α/pMTmCherry was superior to E. coli DH5α/pMTGFP for detecting extractable tetracyclines in soils. This new, fast, easily parallelizable, and cost-effective biosensor method has the potential for measuring extractable concentrations of tetracyclines for a large number of soil samples in large-scale monitoring studies.