The present invention provides a human type 14 replication defective adenovirus vector, and a preparation method for the same, the method comprising: constructing an Ad14 genome into a plasmid, with knocking out E3 and E1 genes of the Ad14 genome, and replacing open reading frames 2, 3, 4, 6, and 6/7 of an E4 gene of the Ad14 genome with corresponding reading frames of an Ad5 genome. The human type 14 replication defective adenovirus vector according to the present invention is potentially applicable in the research and development of a vaccine and a drug against human type 14 adenovirus infection, applicable as a gene vector in the research and development of other pathogen vaccines, and applicable in a biological report and trace system, etc.
