Abstract: Atmospheric pressure low-temperature plasma is a promising tool in biomedicine applications including blood coagulation, bacterial inactivation, sterilization, and cancer treatment, due to its high chemical activity and limited thermal damage. It is of great importance to develop portable plasma sources that are safe to human touch and suitable for outdoor and household operation. In this work, a portable and rechargeable low-temperature plasma spark discharge device (130 mm × 80 mm × 35 mm, 300 g) was designed. The discharge frequency and plume length were optimized by the selection of resistance, capacitance, electrode gap, and ground electrode aperture. Results show that the spark plasma plume is generated with a length of 12 mm and a frequency of 10 Hz at a capacitance of 0.33 μF, resistance of 1 MΩ, electrode gap of 2 mm, and ground electrode aperture of 1.5 mm. Biological tests indicate that the plasma produced by this device contains abundant reactive species, which can be applied in plasma biomedicine, including daily sterilization and wound healing.