The detection of biotin at lower concentration in UV-visible region is difficult because of the low adsorption coefficients. In order to improve this problem, a new migration system, using an exchange reaction between biotin injected and avidin- HABA (2[4’-hydroxyazobenzene] benzoic acid) complex added in running buffer, was developed in the capillary electrophoresis (CE). The use of combination of zwitterionic surfactant (Zwittergent 3–14) and C8 pre-coating capillary enabled this migrating system by suppressing adsorption of avidin for a capillary wall effectively. In this migrating system, the reverse peak height of avidin-biotin complex increased with dependence on concentration of biotin injected. Moreover, the separation of biotin and biocytin was achieved as avidin complex at pH 7 of running buffer. The detection of biotin, contained in a vitamin tablet, was selectively detected avoiding other vitamins by using this CE system. This method enabled detection of only substances having strong affinity with avidin.