To formulate folate receptor (FR)-specific graphene-based electrochemical electrodes, a folic acid (FA) derivative attached with two pyrene molecules on the glutamate tail of FA was synthesized. The resulting pyrene-functionalized FA (FA-Py) presented the spontaneous noncovalent binding on chemically reduced graphene oxides (rGO) through an π-π interaction. Ultrathin morphology, high water-resistance, and preservation of intact FR-specific pteroates from the rGO/FA-Py assembly allow this assembly to be exploited as robust and FR-specific electrochemical electrode materials. The limits of detecting rGO/FA-Py modified electrodes were found to be as low as 3.07 nM in FR concentrations in cyclic voltammetry analysis.