In this study, we developed a microbiological inhibition method for the rapid screening of antibiotics in milk with Geobacillus stearothermophilus ATCC12980 as an indicator bacterium and an easy sample pretreatment. We observed that the limits of detection of the kit for 34 common antibiotic residues in milk, including β-lactams (13), aminoglycosides (6), tetracyclines (4), sulfonamides (6), macrolides (4), lincosamides (1), were lower than or close to the maximum residue limits formulated by the European Union and China. Moreover, the false-positive rate was 1% and the false-negative rates were less than 5%. The ruggedness of the method (the reproducibility of detection capability of different batches of medium) met requirements at determined levels and residual limits. The shelf life of the kit was more than 6 mo at 4°C. Additionally, we observed good correlations between the kit results and ultra-high-performance liquid chromatography-tandem mass spectrometry results for incurred milk (samples taken from animals treated with antibiotics according to the pre-slaughter medication data), which indicated that the kit was reliable for screening antibiotics in incurred samples. In conclusion, the kit has a broad application potential with high sensitivity, specificity, and reproducibility, stability, and reliability, combined with simple operation, low cost, and high-throughput capacity.