A highly sensitive method for determination of berberine is proposed based on the measurements of total internal reflected fluorescence (TIRF) at water/ tetrachloromethane (H(2)O/CCl(4)) interface. In the pH range of 2.6-5.7, the co-adsorption of the berberine with the anionic surfactants such as sodium dodecyl benzene sulfonate (SDBS), sodium dodecylsulfonate (SDS), and sodium lauryl sulfate (SLS) occurs at the H(2)O/CCl(4 )interface, resulting in greatly enhanced TIRF signal characterized by the emission at 526 nm when excited with a 351 nm light beam. The enhanced TIRF intensity is in proportion to the berberine concentration in the range 0.2-10.0x10(-7) mol L(-1). The limit of detection is 1.7x10(-9) mol L(-1) (3sigma). It was found that ions such as Ca(II), Cu(II), Fe(III), Cd(II), Mg(II), Zn(II), Pb(II), and Al(III) can be allowed larger than 1.0x10(-4) mol L(-1). Meanwhile, the organic compounds such as vitamin B, saccharine, and amino acid do not display any effect for the present TIRF method even if they are larger than 1.0x10(-2) mol L(-1)in high concentration levels (larger than 1.0x10(-5) mol L(-1)). The results of determination for synthetic samples were agreement with the desired values, and the ones for tablets were identical with those obtained according to the method of Chinese Pharmacopoeia.