Herein, we report a technique for detecting the fast binding of antibody-peptide inside a capillary. Anti-HA was mixed and interacted with FAM-labeled HA tag (FAM-E-4) inside the capillary. Fluorescence coupled capillary electrophoresis (CE-FL) was employed to measure and record the binding process. The efficiency of the antibody-peptide binding on in-capillary assays was found to be affected by the molar ratio. Furthermore, the stability of anti-HA-FAM-E-4 complex was investigated as well. The results indicated that E(4)YPYDVPDYA (E-4) or TAMRA-E(4)YPYDVPDYA (TAMRA-E-4) had the same binding priorities with anti-HA. The addition of excess E-4 or TAMRA-E-4 could lead to partial dissociation of the complex and take a two-step mechanism including dissociation and association. This method can be applied to detect a wide range of biomolecular interactions.