The invention discloses a method for the in vitro production of active protein, which commonly constructs a target protein gene and the modified protein gene at the downstream of a same promoter in a shuttle vector to generate a fusion gene, then the molecular recombinant method is utilized for recombining an expression frame containing the gene in an adenovirus genome framework, so as to construct a recombinant replicate-defect adenovirus vector, the novel recombinant adenovirus vector containing the fusion gene is introduced into an animal mammary gland after the in vitro auxiliary cell packaging, and the target gene protein with biological activity is obtained from the milk secreted by the animal mammary gland epithelial cells. The active protein generated by applying the method can be directly used as a drug for treatment after separation, purification and concentration, thus greatly reducing the costs of drug research and development, especially the research and the development of human expensive drug protein, and generating huge economic benefits.