•An enrofloxacin detection method based on G-quadruplex structure-switching aptamer have been constructed for the first time.•The novel G-quadruplex structure-switching aptamer retains high affinity and strong specificity for the target.•The detection method has the advantage of sensitivity and a broad detection range.•This proposed sensor was successfully achieved the quantitative analysis of enrofloxacin in various food and environmental samples. Enrofloxacin (ENR) is widely used in the prevention and treatment of animal infectious diseases, so it is necessary to strengthen the residue detection of this drug in animal-derived food and water environments. In this work, for the first time, we engineered assembly a split ENR aptamer into the G-quadruplex (G4) region to form a new aptamer (G4-ENRA) that provides a more sensitive signal-reporting function while retaining target-specific recognition ability of the aptamer. This rational design effectively overcomes the issue of difficulty in identification probe development. Under the optimized conditions, a response range of 0.05–20 µM and limit of detection of 26.7 nM were obtained by directly detecting fluorescence signals, displaying a comparative advantage over the previously reported methods. Moreover, this method demonstrated satisfactory performance for the ENR detection in various real food and environmental samples, with the detection recoveries ranging from 95.87 % to 104.36 %, illustrating promising applicability prospects.