Aptamer affinity capillary electrophoresis-laser-induced fluorescence (CE-LIF) for protein detection takes advantage of aptamers for their ease of synthesis and labeling, small size, and having many negative charges. Its success relies on the high binding affinity of aptamers. One 15-mer DNA aptamer (5'-GGT TGG TGT GGT TGG-3', Apt15) shows desirable specificity for human α-thrombin, an important enzyme with multiple functions in blood. However, Apt15 has weak binding affinity, and the use of Apt15 in affinity CE-LIF analysis remains challenging. Here we reported that extension of Apt15 at the 3'-end with a polyT tail having length of 18 T or longer significantly enhanced its affinity and enabled a well-isolated and stable peak for thrombin-aptamer complex in affinity CE. It was likely that the improvement of binding affinity resulted from double binding, an additional interaction of the polyT tail with thrombin in addition to the Apt15 section binding to thrombin. With dye-labeled Apt15 having a T25 tail, we achieved detection of thrombin at concentrations as low as 0.1 nM by affinity CE-LIF. This aptamer probe specifically bound to human α-thrombin, showing negligible affinity for human β- and γ-thrombin, which are proteolyzed derivatives of human alpha α-thrombin and share similar structure. This strategy of adding a polyT extension also enhanced the binding affinity of anti-immunoglobulin E aptamer in CE-LIF analysis, showing that the affinity enhancement approach is not limited to the thrombin-binding aptamer and has potential for more applications in bioanalysis.