The invention provides a measuring method of mecobalamin tablet related substance. The measuring method comprises the steps of (1) preparing a test sample; (2) preparing reference substance; (3) measuring the content of the related substance by high performance liquid chromatography, wherein the chromatographic conditions are as follows: stationary phase is a chromatographic column taking octadecyl silane bonded silica gel as filler, mobile phase is mixed solution formed by acetonitrile and phosphate buffer solution, the detection wave length is 300-360nm, the sample amount is 20mu l, the column temperature is 30 DEG C, and the flow velocity is 0.5-1.5ml / min; gradient elution is carried out; and (4) calculating the content of all impurities in the test sample. The measuring method of the mecobalamin tablet related substance is high in universality, good in reproducibility, high in degree of separation and accurate in detection result; the methodology validation proves that the measuring method has a stable indicative function, and is capable of rapidly and accurately controlling the limit of all the impurities in mecobalamin tablets, so that the quality can be controlled; furthermore, the measuring method is low in cost, and has good economic benefit and popularization prospect.