Milk safety is a major food safety concern in the United States. The cleanliness of on-farm milking systems directly affects raw milk quality. The generally-accepted four-step procedure for milking system Cleaning-in-Place (CIP) comprises: (1) warm water rinse, (2) alkaline wash, (3) acid wash, and (4) sanitizing rinse prior to the next milking. Electrolyzed oxidizing (EO) water is emerging technology, which generates acidic electrolyzed oxidizing (EO) water and alkaline EO water by electrodialysis of dilute salt solution. Previous studies in our laboratory had shown that EO water can be an alternative for conventional milking system CIP, both on a lab scale milking system and on a commercial dairy farm. Recently, a one-step cleaning has been adopted on an increasing number of dairy farms; it combines the alkaline wash and acid wash cycle together; and this saves chemical expenditure and water usage, energy cost, and time. By mixing acidic EO water with alkaline EO water, a less corrosive but with a high oxidation reduction potential (ORP) blended near neutral EO water can be produced. Therefore, it was proposed in this study that the blended EO water solution can be applied to a lab scale pilot milking system as one-step CIP and the cleaning and sanitization performance between using blended EO water solution and commercial one-step cleaning chemicals are comparable. A response surface method was applied to determine the optimal condition of the cleaning time, the starting temperature of the blended EO water solution and the acidic EO water percentage in the blended EO water solution. The CIP performance comparison among the optimal blended EO water solution and two commercial one-step cleaning chemicals were performed. Results showed that cleaning time of 17 min, a starting temperature of 59 °C and an acidic EO water percentage of 60% in the blended EO water solution (pH of 2.66, ORP of 1109 and chlorine concentration of 48.0 ppm) could achieve the required 100% CIP performance. The CIP performance of using optimal blended EO water solution and the commercial one-step cleaning chemicals showed that the optimal blended EO water solution is comparable to those of the commercial one-step cleaning chemicals. Overall, this study showed that the blended EO water had the potential to be adapted as an alternative for one-step CIP for pilot milking system.