Elevated free fatty acid (FA) levels lead to insulin resistance, hypertension, and microangiopathy, all of which are associated with type 2 diabetes. On the other hand, deficiencies of FA are indicative of certain neurodegenerative diseases, including autism. Thus, free FA levels are a diagnostic indicator for a variety of disorders. Here we describe the use of a commercially available FA binding protein labeled with acrylodan (ADIFAB), which we modified with a ruthenium metal-ligand complex with the intention of creating a low-cost FA sensor. The dual-labeled FA binding protein was used in lifetime-assisted ratiometric sensing (LARS) of oleic acid. For both steady-state and time-resolved luminescence decay experiments, the protein is responsive to oleic acid in the range of 0.02-4.7 microM. The emission at 432 nm, which is associated with the acrylodan occupying the FA binding site, decreases in intensity and red shifts to 505 nm on the addition of oleic acid. The intensities of the 505-nm peak due to the acrylodan displaced from the binding site by FA and of the 610-nm emission peak of ruthenium remained nearly unchanged. Fitting of the fluorescence decay data using the method of least squares revealed three emitting components with lifetimes of approximately 0.60, 4.00, and 370 ns. Fractional intensities of the emitting species indicate that changes in modulation between 2 and 10 MHz on binding of the protein with oleic acid are due mainly to the 4.00-ns component. The 0.60- and 370-ns components are assigned to acrylodan (505 nm) and ruthenium, respectively. Note that because ruthenium has a lifetime that is two orders of magnitude longer than that of acrylodan, the FA measurements were carried out at excitation frequencies lower than what can be done with acrylodan alone. Thus, low-cost instrumentation can be designed for a practical FA sensor without sacrificing the quality of measurements.