Melt-plotted poly (ɛ-caprolactone) (PCL) has been widely applied in various tissue regenerations. However, its hydrophobic nature has hindered its usage in wider tissue engineering applications. In this study, we present the development of a porous and multilayered PCL scaffold, which shows outstanding hydrophilic properties and has a roughened surface consisting of homogeneously distributed nanosized pits. The scaffold was obtained using an innovative oxygen plasma treatment. This technology can induce variable nanoscale surface roughness, which is difficult from traditional plasma treatment. Osteoblast-like cells were cultured on the scaffolds and several cellular responses (cell viability, fluorescence images [live/dead cells, nucleus, and actin cytoskeleton], ALP activity, and calcium mineralization) were assessed for untreated PCL and conventionally plasma-treated PCL scaffolds. The data indicated that an appropriate roughness (654 ± 91 nm) of the PCL scaffold processed with the new plasma treatment induced more advantageous responses for the cells, compared with untreated scaffolds and traditional plasma-treated scaffolds.