With direct evidence that iron is the chemical limitation of phytoplankton growth, particularly in the Southern Ocean, it is increasingly important to develop new tools that provide direct measurement of the bioavailable iron fraction in oceanic waters. Here we report the development of a fluorescence quenching-based siderophore biosensor capable of the in situ measurement of this ultratrace Fe(III) fraction at ambient pH ( approximately 8). Parabactin was extracted from cultures of Paracoccus denitrificans. The purified siderophore was encapsulated within a spin-coated sol-gel thin film, which was subsequently incorporated in a flow cell system. The parabactin biosensor has been fully characterized for the detection of Fe(III) in seawater samples. The biosensor can be regenerated by lowering the pH of the flowing solution, thereby releasing the chelated Fe(III), enabling multiple use. The LOD of the biosensor was determined to be 40 pM, while for an Fe(III) concentration of 1 nM, a reproducibility with a RSD of 6% (n = 10) was obtained. The accuracy of the biosensing system has been determined through analysis of a certified seawater reference sample. Samples from the Atlantic Ocean have been analyzed using the parabactin biosensor providing a concentration vs depth profile for the bioavailable Fe(III) fraction in the 50 pM-1 nM range.