The invention discloses Acipenser sinensis VTG protein antigen and antibody, and a preparation method and an application thereof. The preparation method comprises the following steps: 1, preparation of a recombinant Acipenser sinensis VTG protein antigen: 1) designing an upstream primer and a downstream primer, and amplifying a protein domain truncated sequence; 2) subjecting a pMD-18T-VTG plasmid and a Pet32a (+) expression vector to enzyme digestion by using EcoRI and XhoI, then purifying a target fragment, connecting the pMD-18T-VTG plasmid and the Pet32a (+) expression vector into Pet32a (+)-VTG, converting the Pet32a (+)-VTG into DH5a, coating the DH5a onto an ampicillin containing LB culture medium, and carrying out culturing; 3) converting the Pet32a (+)-VTG plasmid into a BL21(DE3) competent cell, coating the BL21(DE3) competent cell onto an ampicillin containing LB solid culture medium, and carrying out culturing; and 4) subjecting collected mycelium precipitate to resuspension precipitation by using an 8M urea containing protein buffer solution, then carrying out filtering, and collecting a supernatant so as to obtain a recombinant protein; 2, obtainment of an Acipenser sinensis VTG protein antibody through immunization of Japanese rabbit with the recombinant Acipenser sinensis VTG protein antigen; and 3, application of the Acipenser sinensis VTG protein antigen and antibody. The Acipenser sinensis VTG protein antibody and the VTG protein antigen provided by the invention have the following advantages: the immunity is good; a titer can reach 1: 1000; detection results have high positive rate; and the clinical applicability is good.