The invention relates to site-specific integrating expression vectors for therapy Hemophilia B and to methods of preparing them. A vector of the invention contains a human Factor IX gene in a vector constructed using as a chromosome targeting sequence a polynucleotide without any important physiological function-related gene homologous to DNA on the short arms of human group D and human group G chromosomes. The vector of the invention provides high stability of Factor IX expression, high expression efficiency, no immunogenicity and safety in use.