Provided are a human type 55 replication defective adenovirus vector, a method for preparing the same and uses thereof. The human type 55 replication defective adenovirus vector is prepared by the following method: knocking out E1 and E3 genes from Ad55, substituting the open reading frame 6 or the open reading frames 2, 3, 4, 6, and 6/7 of E4 gene in Ad55 genome with the corresponding open reading frames of Ad5 genome. In addition, an exogenous gene expression cassette may also be integrated into the E1 gene region of Ad55.
