Phytoplankton growth in large parts of the world ocean is limited by low availability of dissolved iron (dFe), restricting oceanic uptake of atmospheric CO2. The bioavailability of dFe in seawater is however difficult to appraise since it is bound by a variety of poorly characterized organic ligands. Here, we propose a new approach for evaluating seawater dFe bioavailability based on its uptake rate constant by Fe-limited cultured phytoplankton. We utilized seven phytoplankton species of diverse classes, sizes, and provenances to probe for dFe bioavailability in 12 seawater samples from several ocean basins and depths. All tested phytoplankton acquired organically bound Fe in any given sample at similar rates (after normalizing to cellular surface area), confirming that multiple, Fe-limited phytoplankton species can be used to probe dFe bioavailability in seawater. These phytoplankton-based uptake rate constants allowed us to compare water types, and obtain a grand average estimate of seawater dFe bioavailability. Among water types, dFe bioavailability varied by approximately four-fold, and did not clearly correlate with Fe concentrations or any of the measured Fe speciation parameters. Compared with well-studied Fe complexes, seawater dFe is more available than model siderophore Fe, but less available than inorganic Fe. Exposure of seawater to sunlight, however, significantly enhanced dFe bioavailability. The rate constants established in this work, not only facilitate comparison between water types, but also allow calculation of Fe uptake rates by phytoplankton in the ocean based on measured dFe concentrations. The approach established and verified in this study, opens a new way for determining dFe bioavailability in samples across the ocean, and enables modeling of in situ Fe uptake rates by phytoplankton using dFe concentrations from GEOTRACES datasets.