Progress towards a more circular phosphorus economy necessitates development of innovative water treatment systems which can reversibly remove inorganic phosphate (P) to ultra-low levels (<100 μg L), and subsequently recover the P for reuse. In this study, a novel approach using the high-affinity phosphate binding protein (PBP) as a reusable P bio-adsorbent was investigated. PBP was expressed, extracted, purified and immobilized on NHS-activated Sepharose beads. The resultant PBP beads were saturated with P and exposed to varying pH (pH 4.7 to 12.5) and temperatures (25-45 °C) to induce P release. Increase in temperature from 25 to 45 °C and pH conditions between 4.7 and 8.5 released less than 20% of adsorbed P. However, 62% and 86% of the adsorbed P was released at pH 11.4 and 12.5, respectively. Kinetic experiments showed that P desorption occurred nearly instantaneously (<5 min), regardless of pH conditions, which is advantageous for P recovery. Additionally, no loss in P adsorption or desorption capacity was observed when the PBP beads were exposed to 10 repeated cycles of adsorption/desorption using neutral and high pH (≥12.5) washes, respectively. The highest average P adsorption using the PBP beads was 83 ± 5%, with 89 ± 4.1% average desorption using pH 12.5 washes over 10 wash cycles at room temperature. Thermal shift assay of the PBP showed that the protein was structurally stable after 10 cycles, with statistically similar melting temperatures between pH 4 and 12.5. These results indicate that immobilized high-affinity PBP has the potential to be an effective and reversible bio-adsorbent suitable for P recovery from water/wastewater.