Provided is a recombinant H7N9 subtype avian influenza virus, a marked vaccine and a preparation method thereof. For the recombinant H7N9 subtype avian influenza virus, a strain JD/17 of H7N9 subtype avian influenza virus is used as parent virus and a peptide sequence in HA protein of the strain JD/17 is replaced with a peptide sequence in HA protein of H3 subtype; the strain JD/17 of H7N9 subtype avian influenza virus has a preservation number of CCTCC No. V201862. The results of HA titers, EID50, TCID50 show that the rescued virus maintains similar biological characteristics of parent virus, such as high HA titers and EID50, and chickens immunized with the marked inactivated and emulsified vaccine produce a high level of antibody, and this antibody can be distinguished from antibodies produced by chickens naturally infected with H7N9 subtype avian influenza virus.