Serious healthcare concerns have been raised on the issue of antibiotic residues after overuse, especially by accumulation in the human body through food webs. Here, we report a methodological development for sensitive detection of antibiotics with aptamer conformation cooperated enzyme-assisted SERS (ACCESS) technology. We design and integrate a set of nucleic acid oligos, realizing specific recognition of chloramphenicol (CAP) and efficient exonuclease III-assisted DNA amplification. It features a “signal-on” analysis of CAP with the limit of detection (15 fM), the lowest concentration detectable in the literature. Our method exhibits a high selectivity on the target analyte, free of interference of other potential antibiotic contaminants. The ACCESS assay promises an ultrasensitive and specific detection tool for trace amounts of antibiotic residues in samples of our daily life.