Fluorescent dye Acridine Orange (AO) was indirectly assembled onto the surface of colloidal gold nanoparticles modified quartz and gold wafer, with cysteine as mediation. Strong fluorescence emission was observed from AO self-assembled multilayers. Formation of the self-assembled multilayers was confirmed and primarily characterized by electrochemical and fluorescence methods. The effect of assembling substrate on fluorescence intensity of the film was discussed. The self-assembled multilayers offer a high sensitive interfacial fluorescence sensing of deoxyribonucleic acid with the detection limit of 7.145 x 10(-9) g L(-1) (3 S.D./K), which was five orders of magnitude lower than that of the bulk solution-phase. The self-assembled multilayers film has higher stability and good regeneration capacity. (C) 2010 Elsevier B.V. All rights reserved.