A novel ratiometric fluorescence strategy is developed for specific detection of folic acid (FA) by using 11-mercaptoundecanoic acid protected gold nanoclusters (AuNCs@MUA). In this design, the fluorescence color of the probe can be switched among red, pink, violet and blue by varying the concentration of FA. AuNCs@MUA possesses strong fluorescence peaking at 612 nm (R-signal) and FA exhibits blue emissive auto-fluorescence at 446 nm (B-signal), showing a large emission shift of ∼170 nm. When AuNCs@MUA approaches FA through electrostatic binding, the R-signal decreases while the B-signal increases with titration of FA. Based on the above phenomenon, a radiometric analysis platform is constructed for FA target detection, with a wide linear response range from 0 to 20 μM, and an excellent detection limit of 26 nM. This new ratiometric strategy exhibits low background, and wide signal changes in a low concentration range, which presents obvious advantages over most previous FA detections based on single-responsive fluorescence methods. Furthermore, the proposed method is successfully applied to determine FA in human serum samples.