Enrofloxacin, a widely used fluoroquinolone antibiotic, may be a cause of bacterial drug resistance and is forbidden in poultry. Consequently, a sensitive and rapid method is required for its determination. Aptamers, which are more stable and easily synthesized than antibodies, may serve as alternatives in the development of methods for rapid detection. Six single-strand DNA aptamers binding to enrofloxacin were selected by in vitro selection. Aptamer number 17 showed the highest affinity for enrofloxacin with a dissociation constant of 188 nM and the highest guanine concentration (35%), which was predicted to be crucial for strong affinity of the aptamer to enrofloxacin, and successfully distinguished enrofloxacin from its structure analogs. Using aptamer number 17, a novel chemiluminescent enzyme immunoassay associating with biotin-streptavidin was developed that allowed the determination of enrofloxacin to 2.26 ng/mL. Due to its capability to determine enrofloxacin in bovine milk, this newly selected aptamer may find broad application in food and environmental monitoring.