The invention provides a respiratory syncytial virus pre-fusion F protein and application thereof, and belongs to the technical field of respiratory syncytial virus vaccines. The pre-fusion F proteinis formed in the way that the 106th, 108th and 109th amino acids of a wild-type F protein are mutated from Arg to Asn, the 104th amino acid is mutated from Asn to Cys, the 155th amino acid is mutatedfrom Ser to Cys, or the 58th amino acid is mutated from Thr to Cys, and the 190th amino acid is mutated from Ser to Cys. A first furin cleavage site of the mutant wild-type F protein makes the structure of Pre-F more stable, the expression amount is not decreased, and the pre-fusion epitope of M1-F expression in 293T cells is significantly increased. The second mutation of the amino acid site of the F protein forms a disulfide bond, achieves the more stable pre-fusion epitope compared to the wild type, and is suitable for other strains of human RSV. The protein is suitable for various vaccineforms with the RSV F protein as the antigen, such as nucleic acid vaccines, protein vaccines, vector vaccines and recombinant virus particle vaccines.