Allogeneic red blood cells (RBCs) are commonly used for cardiopulmonary bypass (CPB) circuit priming in congenital heart surgery. While convection-based pre-bypass ultrafiltration (PBUF) corrects acid–base, electrolyte, and metabolite imbalances, its efficacy in removing RBC cytokines/chemokines remains unclear. In a prospective observational study, 22 children (median age: 4.1 months) undergoing congenital heart surgery were enrolled. PBUF of RBC-primed CPB circuits was conducted using bicarbonate-buffered hemofiltration solution. Cytokines/chemokines were quantified in RBC supernatants, CPB priming (before and after PBUF), preoperative patient plasma, and PBUF effluent using Luminex-based multiplex technology. 30 of 50 cytokines were detected in > 50% of RBC supernatants. RBC priming significantly elevated concentrations of 25 cytokines, with 20 further rising after PBUF. At CPB onset, eight mediators (MIF, IL-15, CCL11/Eotaxin, CCL2/MCP-1, VEGF, IL-5, VCAM-1, ICAM-1) exceeded patient plasma concentrations. PBUF filtered cytokines with different efficiencies (0.6–97%). Despite poor filtration or increased concentrations, total mediator load of 42 cytokines decreased significantly (33.3–69.1% of pre-processing levels) after PBUF. In conclusion, PBUF effectively removed multiple cytokines/chemokines released from RBC. Beyond filtration, decrease of total mediator load may be attributed to adsorption to circuit components or rebinding to RBCs. Improved washing techniques may further optimize mediator levels in RBC-primed CPB circuits.