Abstract: Cold plasma was generated using alternating current (26 V) to excite air under atmospheric pressure. The treatment time for each sample group was 0, 1, 2, 3, 4, 5 min, respectively. Plasma components were analyzed by optical emission spectroscopy (OES) and the reactive oxygen species in Klebsiella pneumoniae were investigated by a spectral scanning multimode reader (SSMR). Moreover, membrane potential and membrane permeability of K. pneumoniae cells were monitored by a fluorescence spectrophotometer (FS). The OES results show that there are many ultraviolet (UV) and reactive oxygen species (ROS) when using 26 V power voltage and 3 mm discharge gap. SSMR results indicate the ROS content in K. pneumoniae increases first, and then decreases with discharge time. The cell membrane potential in K. pneumoniae is improved,which leads to the hyperpolarization of cell membrane. FS results show that the cell membrane permeability is enhanced. These results suggest that the barrier discharge plasma at atmospheric pressure has enormous potential in biochemical engineering and technology.