The invention discloses a fluorescent tracing method of an assimilation substance unloading path of a phloem in a pear fruit, comprising the following steps of: preparing a fluorescent solution, introducing the fluorescent solution, sampling, preparing slices, observing and photographing and so on. The invention provides an effective method for researching sugar transportation in the fruit. According to the invention, 5(6)-carboxyfluoresein diacetate CFDA is used as a fluorescent tracing substance and a CFDA solution is introduced into the phloem through a fruit stem part without special equipment and devices, thereby achieving the aim of rapidly and effectively marking the assimilation substance unloading path of the phloem in the development process of the pear fruit. The method provided by the invention has the advantages of no need of the special devices, simplicity in operation, uneasiness of damaging xylems and uneasiness of evaporating the fluorescent solution, no need of special treatments of samples in a fluorescent observation process, and low cost.