The development of sensing systems for the measurement of small molecules is an active area of research. A sensor based approach for the measurement of metabolites can potentially provide the simplicity and portability required for widespread use. Rapid detection and quantitation of small-molecule metabolites can potentially emerge as an effective way to link the metabolite profile to the disease state. Surface plasmon resonance (SPR) combined with molecular recognition elements to deliver high specificity is a sensing platform that has been widely applied for a large range of biomolecules. However, direct detection of small molecules with SPR challenges the refractive index based detection mechanism. The work described here combines a periplasmic binding protein for recognition with target modified gold nanoparticles (AuNPs) in a competitive assay format for folic acid (FA) detection. Specifically, a SPR imaging substrate containing immobilized folate binding protein (FBP) is used to measure the adsorption of FA conjugated AuNPs. The immobilization of the FBP and the binding of the FA conjugated AuNPs are characterized and optimized. It is shown that free FA in solution can be quantitatively measured by competition for the surface binding sites with the functionalized AuNPs. We demonstrate that the dynamic range can be lowered from micromolar to nanomolar by simply decreasing the concentration of FA conjugated AuNPs, thus lowering the limit of detection to 2.9 nM. This type of competitive assay can be applied to a range of small molecules, which paves the way for future multiplexed analysis of metabolites using SPR.