The invention relates to a preparation method of an in-situ glucose detecting membrane based on a fluorescence detection method. Specifically, the detecting membrane is applicable to an implantable in-situ (in vivo) glucose monitoring apparatus for novel minimally invasive, continuous and real-time glucose monitoring. The preparation method comprises the steps of 1) preparing glucose indicator molecules; 2) preparing a material carrier; 3) covalently connecting the glucose indicator molecules prepared in the step 1) to a carrier membrane prepared in the step 2). According to the preparation method of the in-situ glucose detecting membrane based on the fluorescence detection method disclosed by the invention, the glucose indicator molecules are covalently connected to the carrier membrane, so as to guarantee durability; moreover, specific sensitivity and fluorescence activity on glucose are maintained. The detection membrane carrier is free from toxicity or harm and is universal; the carrier can be safely implanted in bodies, so as to achieve a purpose of continuously monitoring glucose in real time.