Electrochemical ozone production (EOP) is an attractive technology for disinfection and sterilization purposes. This work reports a study on the EOP performance of the solid polymer electrolyte (SPE) electrolyzer, including the optimization of electrode configuration and operation conditions. It is proven that the EOP performance is highly affected by electrode configuration. Tests using BDDs with different B/C ratios demonstrate that BDD-4.9 provides more reaction sites and faster electron transfer rate, exhibiting a high electrocatalytic activity for EOP. Regarding electrode thickness, 0.54 mm in thickness is the most suitable for the EOP from the perspective of less power consumption. Moreover, operation conditions were evaluated. It is found that increasing water flow rate is an effective strategy for promoting ozone dissolution and within the present experimental range, the water flow rate of 63 L·h−1 is identified. Meanwhile, through the study of all processes occurring inside the electrolyzer at higher current densities, the optimum current density is determined to be 125 mA·cm−2. Based on these results, the ozone water presents an excellent performance in the killing of E. coli with high inoculum concentrations, indicating the potential application performance in the field of environment.