A label-free ECL aptamer biosensor based on CdS QDs/ACNTs electrode for the sensitive detection of thrombin was fabricated. CdS QDs and Chitosan (CTS) complex films were coated and filled the tubes of ACNTs by carrying out electrodeposition reaction of CTS-CdS QDs on ACNTs electrode, the resulting electrode showed high ECL intensity and good biocompatibility. After aptamer was bound to the film via glutaric dialdehyde, the modified electrode could be used as an ECL aptamer sensor for the thrombin detection. The specific reaction between thrombin and aptamer resulted in the decrease in ECL intensity. The change of the ECL intensity was found to be linear with the logarithm of thrombin concentration in the range from 1.0×10−13 M to 1.0×10−9 M. The ECL aptamer sensor had the advantages of speed, high sensitivity, specificity and stability, which could be a promising technique for protein detection.