Enrofloxacin is widely used in the treatment of animal and plant diseases due to its strong antibacterial activity and broad antibacterial spectrum. However, overuse and even abuse of enrofloxacin can lead to excessive residues in animal and plant food and harm human health through the food chain, so it is crucial to develop efficient methods for enrofloxacin detection. In this work, a novel electrochemical method for enrofloxacin detection is established based on the signal amplification strategy designed by collaborating terminal deoxynucleotidyl transferase (TdT) with G-quadruplex ribozyme. The binding of target enrofloxacin with specific aptamer triggers the extension reaction of TdT on the electrode surface, resulting in the formation of G-quadruplex ribozyme nanowires, which can play horseradish peroxidase activity to catalyze signal amplification, finally achieving highly sensitive and specific detection of enrofloxacin. The linear detection range of enrofloxacin is 0.5 ~ 50 ng/mL, and the limit of detection is as low as 0.043 ng/mL. In addition, the label free electrochemical biosensor is simple, fast and low cost, and has been successfully applied to the analysis of real food samples, showing good application potential.