The invention discloses a method for constructing an adenovirus core protein genetic marker vector, which comprises the following steps of: constructing a shuttle vector for introducing an enzyme cutting site to the 3' end of an adenovirus genome core protein DNA sequence, introducing an adenovirus skeleton vector of the enzyme cutting site, constructing a marker gene for marking the core proteinshuttle vector, constructing a marker gene for marking a core protein adenovirus plasmid vector, and packing and purifying marker gene marked core protein adenoviruses. The invention adopts a new method for marking the adenovirus core protein, the method realizes real genetic-level fluorescent mark of the adenovirus core protein, and the marking method deletes the original core protein gene in the adenovirus genome, so the method for marking the adenovirus core protein can obtain virus granules completely marked by the fluorescent protein.