The serum albumin is the most abundant protein in blood plasma and the iron is essential for many cellular processes. However, the interaction between Fe3+ and haem-free serum albumin remains unclear. Here we provide evidence for the fact that haem-free BSA possesses one specific Fe3+-binding site. The binding of Fe3+ to BSA results in a significant quenching of the Trp fluorescence of BSA. The average apparent dissociation constant value for the interaction of Fe3+ and BSA is 3.46 × 10−8 ± 3 × 10−10 M at 37 °C and 3.30 × 10−8 ± 5 × 10−10 M at 25 °C, respectively, as determined by fluorescence titration. Addition of 50 μM Fe2+ to 1 μM BSA results in an obvious hysteretic effect on the fluorescence of BSA. The time-dependent fluorescence quenching of BSA by Fe2+ is not caused by the Fe2+-induced conformational change of BSA, but the oxygen-dependent oxidation of Fe2+ to Fe3+. Fe2+ undergoes an oxygen-dependent oxidation to Fe3+ under aerobic conditions, which is accelerated by the interaction of BSA with Fe3+ and extensively inhibited under anaerobic conditions. The results suggest that BSA may take part in non-transferrin bound iron transfer.