A simple and sensitive electrochemical sensor was developed for the detection of folic acid (FA) based on gold nanoparticles (AuNPs) dispersed on exfoliated MoS₂ (2D-MoS₂) nanosheets by a simple impregnation method. The prepared 2D-MoS₂ and AuNPs-MoS₂ samples were characterized by scanning electron microscopy (SEM-EDX), UV–vis, and Raman spectroscopy. Screen-printed carbon electrodes (SPCE) were modified by the 2D-MoS₂ and AuNPs-MoS₂ nanosheets and their electrochemical characteristics were investigated by means of electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques and tested in the electroanalytical determination of folic acid (FA). The modified 2D-MoS₂/SPCE was found to promote the FA electrooxidation process compared to bare SPCE. Instead, with the AuNPs-MoS₂/SPCE sensor a different sensing mechanism, based on the inhibition of the Au NPs redox peaks current, has been found to be more effective. This behavior was due to the strong binding of FA on AuNPs, as probed by in situ ATR-FTIR and SERS measurements. The decrease of the anodic peak current of Au NPs with the concentration of FA provided an indirect signal which has been used here for detecting FA.