Bio-adsorption using high-affinity phosphate-binding proteins (PBP) has demonstrated effective phosphorus removal and recovery in batch-scale tests. Subsequent optimization of design and performance of fixed-bed column systems is essential for scaling up and implementation. Here, continuous-flow fixed-bed column tests were used to investigate the adsorption of inorganic phosphate (orthophosphate, Pi) using phosphate-binding proteins immobilized on resin (PBP-NHS) targeting Pi removal to ultra-low levels followed by recovery. Time to breakthrough decreased with higher influent Pi concentration, smaller bed volume, and higher influent flow rates. The Thomas and Yoon-Nelson breakthrough models adequately described PBP-NHS resin performance with a correlation coefficient of R2 > 0.95. The sharp S-shape of the breakthrough curves for both Pi-only solution and multi-ion solution indicated highly favorable and selective separation of Pi using PBP-NHS resin, beyond that achieved using LayneRT™, a commercial ion exchange resin. The Pi adsorption capacity of the PBP-NHS column was unaffected by competing anions, whereas capacity of the LayneRT™ column dropped by 20%. Tertiary wastewater effluent was also successfully treated in PBP-NHS column tests with a typical S-shaped breakthrough curve. Operating the fixed-bed column in multi-cycle mode evidenced the reusability of PBP-NHS resin with no significant decline in column performance. The results of this study contribute to efforts to scale up designs of PBP-NHS adsorption systems.