A novel coaxial surface dielectric barrier discharge (SDBD) non-thermal atmospheric pressure plasma device, driven by a 35.7-kHz DC pulse and adjustable by a pulse-width modulation, was developed in this study and preliminarily tested for its killing effects of a cancer cell type. This study was divided into three phases, namely, air phase, liquid phase, and cell phase. First, the electrical characteristics and emissions were examined. Two-beam UV-LED absorption spectroscopy was also newly developed to measure the absolute hydroxyl radical (OH⋅) density in the filamentary discharge. Then, the effects of energy doses and treatment durations on three types of liquids and on the colorectal adenocarcinoma cell, SW620, were examined. From Lissajous figures (Q–V plot), the developed SDBD possesses the maximum power density and energy dose of 0.33 ± 0.05 W/cm2 and 19.5 ± 3.00 J/cm2, respectively, when the voltage was set at 3.44 kV and the power at 115 mW. From two-beam UV-LED absorption spectroscopy results, the OH⋅ density increased by 0.32, 0.58, and 0.86 × 1019 m−3, with operational powers of 29, 58, and 115 mW, respectively, within 1-min treatment. In liquid phases, the plasma device can increase the concentrations of H2O2 and NO2− in a time-dependent manner. Finally, cell-phase studies, including the examination of the cell morphology, cell viability, and gene expression of the SW620 cell, show that the device can time-dependently induce the mortality of the SW620 cell, relevant to the up-regulation of the Bax/Bcl-2 expression ratio. Taken together, this novel SDBD plasma device shows potential as another alternative for cancer treatment, although further modification is required.