The invention relates to a chemically synthesized beta-1,4-galactosyltransferase I (beta-1,4-GalT I) substrate fluorescent molecular probe targeting to tumor cells, and an intermediate and a preparation method thereof. The chemical structure of the beta-1,4-GalT I substrate fluorescent molecular probe is shown in the description, and Ac in the formula represents an acetyl group. Tumor cell strains are screened to obtain high-expression beta-1,4-GalT I adopted as a target, a 8-difluoromethyl-7-hydroxycoumarin-beta-acetylglucoside specific substrate is designed and synthesized based on an ASPs strategy, the galactosyl group of a donor UDP-Gal is transferred to the substrate through using the transglycosylation of beta-1,4-GalT I in order to generate a lactosyl group, aglucones are released in the glycosidase hydrolysis process and undergo an elimination reaction to form a quinonylmethyl compound with high reaction activity, and the quinonylmethyl compound is covalently combined with the nucleophilic substrate nearby the enzyme to finally complete targeting fluorescent labeling process of the tumor cells, so early diagnosis of the tumors is realized.