The development of an effective sensing platform is necessary for highly selective, sensitive, and rapid screening of specific antibiotics. In this study, graphene oxide was adopted to efficiently quench the fluorescence emission of three different fluorophores. Graphene oxide quenched fluorescently modified aptamers with efficiencies of 94.36%, 93.94%, and 96.97% for Cy3, FAM, and Cy5, respectively. An endonuclease-assisted cyclic enzymatic signal amplification method was used for sensitive detection of antibiotics (e.g., sulfadimethoxine, kanamycin, and ampicillin), resulting in a 5-fold increased signal compared to that of unamplified fluorescent methods. The aptasensor rapidly detected antibiotics in solution with limit of detection values of 1.997, 2.664, and 2.337 ng/mL for sulfadimethoxine, kanamycin, and ampicillin, respectively. In addition, antibiotics dissolved in milk were efficiently detected with similar sensitivities. These results indicate that the aptasensor offers high specificity for each antibiotic and enables simultaneous and multicolor sensing for rapid screening of multiple antibiotics.