Antibiotic pharmaceutical wastewater is difficult to be treated in wastewater treatment field. The study of interaction mechanism between antibiotic and microorganism is of great significance for solving antibiotic wastewater treatment problem. As Raman spectroscopy offers a nondestructive, noninvasive, and water-insensitive test tool, it has been more and more widely applied in the study of biology and medicine. This work isolated 4 strains of antibiotic-resistant bacteria from activated sludge taken from sewage treatment reactor by using streak plate method and spread plate method, and the drug degradation effect of these strains is assessed, which shows the highest degradation rate reached 45.44%. Besides, sequence of these strains’ 16S rRNA gene was analyzed to identify them, and the result shows that these strains belong to Aeromonas sp., Bacillus sp. and Rhodococcus sp.. Raman spectroscopies of these stains under different conditions were taken to investigate the best test condition of bacteria’s Raman spectroscopy, and the result shows that CaF2 can be the best substrate. Then the Raman spectroscopies of these strains were obtained and the difference among them is analyzed by using principal component analysis, and three principal components were extracted, of which the accumulated load reached 83.9%. Among all the variables, 748, 1 003, 1 126 cm-1 belong to the first principal component, 1 661, 1 448 cm-1 belong to the second, and 855 cm-1 belongs to the third. This research characterized biological macromolecules of stains screened from activated sludge by Raman spectroscopy and well distinguished them, proving the reliability and superiority of Raman spectroscopy for applying to mechanism study of microorganism interaction in wastewater treatment.