We present a surface-enhanced Raman scattering (SERS) substrate featured by large-scale homogeneously distributed Ag nanoparticles (Ag-NPs) with sub-10 nm gaps assembled on a two-layered honeycomb-like TiO(2) film. The two-layered honeycomb-like TiO(2) film was achieved by a two-step anodization of pure Ti foil, with its upper layer consisting of hexagonally arranged shallow nano-bowls of 160 nm in diameter, and the lower layer consisting of arrays of about fifty vertically aligned sub-20 nm diameter nanopores. The shallow nano-bowls in the upper layer divide the whole TiO(2) film into regularly arranged arrays of uniform hexagonal nano-cells, leading to a similar distribution pattern for the ion-sputtered Ag-NPs in each nano-cell. The lower layer with sub-20 nm diameter nanopores prevents the aggregation of the sputtered Ag-NPs, so that the Ag-NPs can get much closer with gaps in the sub-10 nm range. Therefore, large-scale high-density and quasi-ordered sub-10 nm gaps between the adjacent Ag-NPs were achieved, which ensures homogeneously distributed 'hot spots' over a large area for the SERS effect. Moreover, the honeycomb-like structure can also facilitate the capture of target analyte molecules. As expected, the SERS substrate exhibits an excellent SERS effect with high sensitivity and reproducibility. As an example, the SERS substrate was utilized to detect polychlorinated biphenyls (PCBs, a kind of persistent organic pollutants as global environmental hazard) such as 3,3',4,4'-pentachlorobiphenyl (PCB-77) with concentrations down to 10(-9) M. Therefore the large-scale Ag-NPs with sub-10 nm gaps assembled on the two-layered honeycomb-like TiO (2) film have potentials in SERS-based rapid trace detection of PCBs.