Pseudomonas fluorescens cultures produce fluorescent siderophores. By utilizing optimal conditions for maximizing siderophore production in shake flask cultures of P. fluorescens, we report successful characterization of the culture broth supernatant as a robust ferric ions biosensor. For characterizing the ferric ions biosensor, we tested the effects of pH, buffers, different ferric salts and possible interference by ferrous ions under different solution conditions. We find that the biosensor is very specific to ferric ions only with sensitivity to concentrations as low as 10 mu M. Further, the response time of the biosensor is the shortest (similar to 5 min or smaller) for citrate as the accompanying anion with ferric ions. While the response time is longer than that expected of normal biosensors, it is well compensated by the simplicity and economics of the biosensor production. Extremely low standard deviations in several experimental repeats also highlight the robustness of the ferric ions biosensor. Most importantly, the biosensor is extremely easy to use due to its straightforward spectrophotometric applications. We also show the utility of the biosensor with the high resolution technique of fluorescence microscopy. Finally, we report a novel mechanistic finding that siderophores present in the culture broth supernatants have two distinct optically active sites on them, which can be monitored independently in presence or absence of ferric ions.