We alternately deposited negatively charged Ag-(3-mercaptopropionic acid) (Ag-MPA) sol and positively charged poly-(diallyldimethylammonium) (PDDA) on gold substrate modified with 4-aminothiophenol (4-ATP), through electrostatic layer-by-layer (LBL) self-assembly. We characterized the prepared three-dimensional Ag/PDDA multilayer films by surface plasmon resonance (SPR) and atomic force microscope (AFM). The thickness of each film in the multilayer films, the deposition effect of Ag nanoparticles, and the processing of DNA adsorption are characterized by SPR. AFM characterization shows that DNA/3(PDDA/Ag)/4-ATP composite is uniformly and firmly distributed on the surface of gold films. Compared with other sensors, gentamicin could be highly sensitively measured by DNA/3(PDDA/Ag)/4-ATP/Au sensor. There is a good linear relationship in the concentration range of 5x10(-8) to 1x10(-4) mol/L. The linear equation is found to be Delta theta(SPR) = 1.3521x10(-5)c + 0.08641 (the correlation coefficient is 0.9983) with detection limit of 1x10(-9) mol/L. Since such LBL assembly film is simple to prepare, the work described here provides an effective method for studying small molecule drugs on SPR. (C) 2010 Elsevier B.V. All rights reserved.