The invention discloses human adenovirus antigen epitope chimeric protein as well as preparation and application thereof, and relates to fields of gene engineering techniques, vaccines and diagnostic reagents. The amino acid sequences of hexon protein of type 3, type 7, type 11, type 14 and type 55 of adenovirus are analyzed through computer analysis, protein fragments with good antigenicity can be screened respectively, the fragments are connected by using two glycine and one serine, then chimeric protein with multiple antigen fragments in serial connection can be formed, pronucleus preferred codons are selected and interpreted into corresponding nucleotide sequences, and full-length genes can be synthesized in a chemical manner. The chimeric protein can be obtained through expression purification by using a gene engineering technique, and the chimeric protein comprises 363 amino acids in whole length. The expressed chimeric protein can be applied to vaccine research, HAdV antibody or antigen detection and the like, and is related to fields such as gene engineering techniques, vaccines and diagnostic reagents.