The invention discloses a double-staining method for bovine in vitro fertilized blastocysts, which comprises treating the bovine in vitro fertilized blastocysts cultivated to 7-9 days with 0.5% pronase for 55 seconds, and then washing them in PBS for 2 to 3 times. Stain in 100µg / ml PI (1%TritonX‑100 / PBS) for 20s, wash in PBS for 2-3 times, transfer to 45µg / ml Hoechst33342 for staining for 50s, wash in PBS for 2-3 times, and press. After pressing, the tablets were observed and photographed under an inverted fluorescent microscope under ultraviolet light. The nuclei of the blastocyst inner cell mass (ICM) are blue, and the nuclei of trophoblast (TE) cells are red. The staining method can be completed in 3 minutes, while the previous report required at least 47 minutes. Therefore, the time required for double staining of blastocysts is greatly reduced, and the efficiency is improved, so that the quality of bovine blastocysts can be quickly evaluated.