Antibiotics, which can be used as veterinary drugs, are widely used in the prevention and treatment of infectious diseases for animals. However, overuse of antibiotics had caused serious problems on food contamination and human harm. For control such public issues, several of techniques have been in recent years. Ratiometric fluorescent (RF) technique, as one of the most promising strategies for quantitatively evaluated analytes, had been extensively developed for the readily measurements on the two different fluorescent emission intensities. In this review, the construction strategies for recent RF sensors will be mainly focused on. Meanwhile, the recent advances and new tendencies for detection of antibiotics based on RF technique shall be introduced. Finally, outlooks on the opportunities and challenges for quantitative fluorescence sensing on antibiotics will be summarized.