The present invention relates to the field of biotechnology and specifically discloses a replication-deficient type human type 7 adenovirus and a preparation method and an application thereof. In thereplication-deficient type human type 7 adenovirus, E1 gene and E3 gene are deleted, E4 gene open reading frames 2, 3, 4, 6 and 6 / 7 are replaced into a corresponding reading frame of Ad5 genome, and aE1 gene region can integrate foreign gene expression box. The replication-deficient type 7 adenovirus can be successfully rescued and mass-produced in HEK293, but does not have replication ability inhuman normal cell lines; and after the replication-deficient type 7 adenovirus carrying foreign genes infects cells, efficient expression of the foreign genes can be realized. The replication-deficient type 7 adenovirus can potentially be applied in development of preventive vaccines against human type 7 adenovirus infection; neutralizing antibodies and drug screening against human type 7 adenovirus infection; application as a gene carrier for development of vaccines for other pathogens; and report tracking systems for biological research, etc.