The invention discloses a preparation method and an application of a tissue slice for observing temporal-spatial distribution of early embryo development in vivo. The preparation method comprises the following steps that 4% paraformaldehyde fixing, upward gradient ethanol dehydration, wax dipping, embedding, serial section, baking, dewaxing and downward gradient ethanol rehydration are performed in sequence on oviducts or uterine tissues which contain mice embryos in every period, and finally, after haematoxylin-eosin staining is performed on the tissues in the slice, neutral gum is used for sealing the slice, or after immunofluorescence histochemical staining is performed on the slice, a fluorescence resistant quenching sealing agent is used for sealing the slice. The tissue slice disclosed by the invention can be used for manufacturing a map of early mice embryo development and detecting the expression of Crb3 in the mice embryos in every period of development in vivo. The preparation method has the advantages that positions of all organs in the embryos can be relatively fixed, so that the position change of embryo cells in a genital tract and the continuity of embryo development can be conveniently observed, the structure is clear, and the tissue slice is convenient to store.