Catfish ranked 8th among seafoods consumed per capita in the United States (U.S.) in 2019. As a major catfish product, catfish fillets are often chilled or frozen for storage after filleting and trimming operations. When they are removed from storage for further processing, manual interference is required to separate and flatten all the catfish fillets that otherwise could agglomerate together. This is necessary for further processing operations such as breading and individual quick-freezing. Because of the shortage of labor force and rising labor costs, there is an urgent need to reduce labor dependence by developing automated singulation methods for catfish fillets manufacturing, to enhance the profitability of the U.S. catfish industry. This study presents a novel singulation system that enables separating catfish fillets by utilizing water buoyancy and underwater streams. Several operation parameters have been investigated and optimized to achieve better singulation performance and production rate. The continuous production mode with operation rules is defined to keep the total amount being processed in an appropriate range for maximizing the singulation effect and production rate. This study will be beneficial for developing more effective next-generation singulation technologies for catfish and other fish species.