The invention discloses a chromatographic detection method for simultaneously analyzing ammonium ions, amino acids and biogenic amine in tobacco leaves. Thechromatographic detection method comprises the following steps of A, extracting the ammonium ions, amino acids and biogenic amines in the tobacco leaves; B, deriving the ammonium ions, amino acids and biogenic amines in the tobacco leaves; C, performing the chromatographic analysis on the derived products of ammonium ions, amino acids and biogenic amines in the tobacco leaves; D, quantitatively analyzing the ammonium ions, amino acids and biogenic amines in the tobacco leaves. The chromatographic detection method has the advantagesthattheammonium ions, 25 types of amino acids and 8 types of biogenic amines in tobacco leaves can be simultaneously measured by microwave-assisted extraction, ethoxy methylene malonic diethyl esterderiving, ultra-high performance liquid chromatography and ultraviolet detectors; the simplicity and rapidityare realized, the sensitivity is high, thestability is good, and the like; the detection flux and data accuracy of the sample can be improved.