Glycidol‐modified polyethylenimine (PEI‐OH)‐capped carbon dots (PEI‐OH‐CDs) were prepared and applied for selectively and sensitively detecting folic acid in food samples. PEI‐OH‐CDs were firstly synthesized by a one‐pot hydrothermal method using PEI‐OH and citric acid as precursors, and the transmission electron microscopy characterization reveals that the PEI‐OH‐CDs were of spherical shape with diameters of 2–3 nm and well dispersed in solution. The PEI‐OH‐CDs have high fluorescence and exhibit ultrastrong stability against pH, ion strength, and UV light irradiation. In a wide pH range higher than pH 6, the fluorescence of PEI‐OH‐CDs can be effectively quenched by folic acid through static quenching process and inner filter effect but is not affected by many typical chemicals including cations, anions, amino acids, and vitamins. A good linear relationship was found between the F0/F and the concentration of folic acid in the range from 0.1 to 100 μM, and the detection limit of folic acid is 0.07 μM. The PEI‐OH‐CDs were successfully applied for sensitive determination of folic acid in orange juice, fresh pear, grape spinach, and celery samples at pH 7.2, which is helpful to the stability of folic acid and provides a new strategy for accuracy detection of folic acid in food samples.