In this research work, ultrasensitive and reliable ratiometric sensor was designed for assay of ciprofloxacin (CIPRO). The platform consists of thiolate-protected gold nanoclusters (GSH@AuNCs) with high fluorescence quantum yield and long-term stability. In the existence of lanthanum (La3+) ion, the GSH@AuNCs emission was sharply raised owing to the formation of La3+/GSH@AuNCs system accompanied with aggregation-induced emission enhancement (AIEE). Addition of CIPRO, a good La3+ ion coordinator, decreased the fluorescence emission of La3+/GSH@AuNCs system at 610 nm significantly as a result of disaggregation caused by the removal of La3+ ion from GSH@AuNCs surface. Furthermore, the blue emission of La3+-CIPRO coordination complex appears at 440 nm. Under optimized conditions, the fluorescence ratios (F440/F610) were linearly increased with increasing the CIPRO amount within the range of 0.003–200 µM with a limit of detection equal to 0.2 nM (S/N = 3). The as-fabricated La3+/GSH@AuNCs system possesses outstanding reliability and sensitivity for the detection of CIPRO. The system was effectively used to assay CIPRO in human urine and milk specimens with recoveries % of 97.6–102.3 % and 96.7–105.7 % for urine and milk samples, respectively and RSD % did not exceed 2.5 %, suggesting the accuracy of the method.