The invention discloses a molecular biological identification method for I type duck hepatitis virus, which comprises the steps of treating samples to be tested, preparing RNA of the samples, obtaining cDNA of the samples by reverse transcription, identifying I type duck hepatitis virus by the fluorescent quantitive RT-PCR method or the nested PCR method, etc. The molecular biological identification method has the advantages of fast diagnosis, high sensitivity and wide applied range, can be used for identifying DHV-1 from the tissue samples, cotton swabs, ducks or chicken embryo allantoic liquid or cell cultures.