A novel electrochemical aptasensing transducer platform has been developed based on carbon nanotube (CNT)-V2O5-chitosan (CS) nanocomposites modified screen printed carbon electrode (SPCE) for the detection of ciprofloxacin. Using CNT-V2O5-CS/SPCE as the basal electrode transducer platform for the immobilization of the single-stranded DNA aptamer, and the electrochemical impedance spectroscopy (EIS) was selected for quantitative detection of ciprofloxacin (CIP). This aptasensor combines the biocompatibility of V2O5 nanoparticles, the efficient electron transfer capability of multiwalled CNTs, the effective film-forming strength of CS and the portability of SPCE. Under optimized experimental parameters, the developed aptasensor exhibited a dynamic range from 0.5 to 64.0 ng mL−1, where the linearity was between 0.5 to 8.0 ng mL−1 and the limit of detection (LOD) was 0.5 ng mL−1 of CIP. Furthermore, it showed good selectivity to CIP. For practical application, the aptasensor performance was verified in spiked milk samples and the acceptable recovery percentage of 94.50-97.87% (%RSD = 4.38, n = 3) was obtained. © 2018 Elsevier B.V.