The binding reaction of reduced graphene oxide-silver nanocomposites (rGO-AgNCs) with calf thymus single-stranded DNA (ssDNA) was studied by ultraviolet-visible absorption, fluorescence spectroscopy and circular dichroism (CD), using berberine hemisulphate (BR) dye as a fluorescence probe. The absorbance of ssDNA increases, but the fluorescence intensity is quenched with the addition of rGO-AgNCs. The binding of rGO-AgNCs with ssDNA was able to increase the quenching effects of BR and ssDNA, and induce the changes in CD spectra. All of the evidence indicated that there was a relatively strong interaction between ssDNA and rGO-AgNCs. The data obtained from fluorescence experiments revealed that the quenching process of ssDNA caused by rGO-AgNCs is primarily due to complex formation, i.e. static quenching. The increasing trend of the binding equilibrium constant (Ka) with rising temperature indicated that the binding process was an endothermic reaction. The calculated thermodynamic parameters showed that the binding process was thermodynamically spontaneous, and hydrophobic association played predominant roles in the binding of ssDNA to the surface of rGO-AgNCs.