Years ago, we proposed the ciliate Euplotes balteatus as a candidate agent for harmful algal bloom (HAB) mitigation, as it efficiently grazes multiple HAB species, notably toxic dinoflagellates such as Karenia mikimotoi and Alexandrium minutum. In this study, to enable scalable propagation of E. balteatus for field application, prey combinations were evaluated using a response surface methodology (RSM)-optimized design, and a self-sustaining predator–prey system with rapid-startup capability was established using an Escherichia coli–Chaetoceros muelleri combination. Population dynamics analyses showed that E. balteatus rapidly initiates system assembly on E. coli and transitions to a C. muelleri-supported regime exhibiting long-term oscillatory dynamics. Experimental assays in monocultures and field-collected seawater demonstrated that the HAB mitigation efficiency of the self-sustaining system was comparable to that of E. balteatus alone. These results suggest that coupling E. balteatus with a renewable prey source provides a promising strategy for biological control of harmful algal blooms.