The invention belongs to the technical field of animal gene engineering, and in particular relates to clone of an HIF3alpha gene segment of a cattle excellent superovulation character related gene by utilizing a specific primer and an application thereof in cattle marker-assisted selection. The length of the nucleotide sequence of the acquired HIF3alpha gene segment is 379bp; an A->G base mutation exists in the 278thbp of the segment, which causes generation of Alw44I-RFLP restriction fragment polymorphism; and the mutation site is used as a molecular marker, and is detected by using restriction enzyme Alw44I, and the detection result and the cattle excellent superovulation character are related. Analysis results prove that the superovulation characters of individuals with different gene types are remarkably different. The identification method has the characteristics that partial segment of the HIF3alpha gene related to the cattle excellent superovulation character is acquired by utilizing the specific primer, and the segment is subjected to polymorphism analysis by utilizing a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method, so that a molecular marker and an application thereof are provided to cattle marker-assisted selection.