The invention belongs to the field of inspection and quarantine technology. Specifically, the invention is a nucleic acid detection reagent kit for synchronous discriminating and diagnosing avian influenza virus and newcastle disease virus. The detection reagents of the reagent kit include extraction reagent for extracting virus by silicon gel absorption column method, detection amplification reagent for detecting nucleic acid by RT-PCR Taq Man fluorescent probe method, and pretreatment liquid for solid tissue specimen for extracting virus RNA. Further, the invention employs in vitro transcription RNA as a positive contrast of the reagent kit. The reagent kit can rapidly and synchronously discriminate and diagnose avian influenza virus and newcastle disease virus which are highly infectious among avian plagues and have similar symptom, determine current major prevalent subtypes, such as H5, H7, H9, etc., and discriminate whether a infection source is an avian influenza having high pathogenicity, non-pathogenic avian influenza or mildly pathogenic avian influenza to human. The reagent kit is suitable for livestock and veterinarian station, import and export inspection and quarantine bureau, as well as other laboratories, and can be used for large-scale detection of influenza and epidemic surveillance.