Losses in energy storage systems (ESSs) are considered operational costs and it is critical to improve efficiency in order to expand their use. We proposed a method of improving efficiency through the operation algorithm of an ESS, consisting of multiple energy storage units (ESUs). Since the ESS used for frequency adjustment maintains a fast response characteristics, we have created a lookup table of efficient ESU operating combinations using genetic algorithms in advance to maintain a fast response time. In addition, the charge/discharge energy balance between the plurality of ESUs is maintained, and the deterioration is prevented from being concentrated in some ESUs. The proposed algorithm was reviewed based on three case studies. The first case study examined whether the lookup table obtained using the genetic algorithm well identified the optimal solution. The second compared the system that optimally distributes the output to ESUs to achieve ESS efficiency for frequency regulation with the system that uniformly distributes the output to ESUs. The third analyzed the impact of efficiency enhancement on the operation of an ESS for frequency regulation through a one-month simulation. The findings confirmed that when the entire system output is optimally allocated to the ESUs, the system’s economic efficiency can be improved by reducing the loss while maintaining the accumulative charge/discharge balance.