Objective To develop a detection method for antibiotic residues in laboratory animal serum, which ensure the accuracy of the results of the microbiological quality monitoring. Methods Antibiotic residues of 1640 laboratory animal sera were test by microbial inhibition assay of Bacillus stearothermophilus. And the results compared with the antibiotic detection kit Premi@Test. Results Antibiotic residues were positive in sera of mice, rats, guinea pigs, rabbits and dogs, the positive rate of 27.2%, 16.4%, 39.9%, 23.7% and 47.6%, respectively. The results were consistent with Premi@Test（X2=16.680，P>0.05). Sera antibiotic residues and DHL agar colony growth had certain correlation(X2=9.939, P <0.05). Conclusions The microbial inhibition method of Bacillus stearothermophilus used as indicator is not only capable of detecting antibiotic residues in animal sera, low cost, simple operation, but also has a good sensitivity and repeatability. This study provides a basis for detection of antibiotic residues in the laboratory animals, and makes up for the blank in the quality testing of laboratory animals.