Antibiotics are one of the most widely used types of drugs in pharmaceutics. However, efficiency of these drugs has decreased recently owing to the threat of antibiotic resistance. One of the important factors causing antibiotic resistance is the excessive use of antibacterials in animals. Therefore, detection of antibiotics in foods of animal origin is crucial. The aim of this study was to develop a novel whole-cell based bioassay to be used for detection of some antibiotics. Green fluorescent protein (GFP)-expressing Escherichia coli cells were used as a recognition agent, and antibiotic detection was carried out by pursuing the inhibition rate of fluorescence intensity as a result of the inhibition of viable cells by the time of progress. The performance of bioassay was tested for different antibiotics, and the obtained results showed that the developed method can be used successfully for detection of ampicillin, benzylpenicillin, gentamicin, neomycin, and tetracycline with the limit of detection (LOD) values of 3.33, 0.29, 28.00, 618.36, and 33.17 mu g/L, respectively. The assay was also tested with antibiotic spiked milk samples (skimmed UHT, full-fat UHT, and whole raw milk). According to obtained recovery values, developed method was successful for all samples. The precision and bias values of the method were found between the range of 1.30% to 7.54% and -8.00% to 0.64%, respectively. The developed method, which is inexpensive and simple with detection limits in line with the regulatory limits, is promising for use in milk quality monitoring. Method has potential to be used as a screening method after comprehensive validation. Practical Application This method could be used in animal husbandry to check whether the antibiotic prescribed for the treatment of sick animals is still present in their milk as residual. For dairy industry, detection of residual antibiotics in milk is crucial because of their inhibition effects on the fermentation processes. Therefore, the proposed method can be used for routine analysis of raw milk reception in dairy industries. In addition, it is considered to have a wide range of applications for all foods.