Dendrimer multilayers on gold substrates prepared via layer-by-layer (LbL) assembly technique were characterized and used as substrates for DNA immobilization/hybridization. The multilayers, built using alternately polycationic and polyanionic phosphorus dendrimers of generation 4, were studied by surface plasmon resonance (SPR) spectroscopy. By varying the concentration of NaCl, the optimized optical thickness of a single dendrimer layer (about 4.5 nm) was achieved. Using the multilayers as the substrate, a high loading of DNA probes was obtained through covalent coupling of probe DNA on dendrimer multilayer film. The following hybridization of Cy5-dye labeled complementary target DNA with immobilized probe DNA was detected by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). The limit of detection of target DNA upon hybridization reached 50 pM and 30 pM on 1 bilayer and 4 bilayers, respectively. The phosphorus dendrimer multilayer Films display high stability during repeated regeneration and hybridization cycles. The sensitive platforms based on dendrimer multilayers deposited in the presence of NaCl make them attractive candidates for application in DNA sensing.