Deformed wing virus (DWV) is a serious pathogen of the honeybee, Apis mellifera L., vectored by the parasitic mite Varroa destructor. The virus is associated with wing deformity in symptomatic bees, and premature death and reduced colony performance in asymptomatic bees. In present study a novel micro PCR-based detection method, termed as ultra-rapid real-time PCR (UR-RT PCR), was developed for the fast and quantitative detection of DWV in honeybee. A specific detection primer set (DWV-UR-F3/R3) was used for the amplification of an unique 133-bp DNA fragment of DWV with a rapid real -time PCR system, GenSpector® TMC-1000, which proceed the cycling with fast heating and cooling rates and a small reaction volume. We showed that this method is able to detect DWV with DNA conditions, artificial recombinant DNA, pBX-DWV479 as well as with virus-infected honeybee samples. In application to a DWV-infected honey bee, the minimum detection time was 8 min 50 seconds under 30 cycles and 10min 11 seconds including melting temperature analysis. This optimizing detection method is one of the fastest real-time PCR-based diagnostic tools and is available to be applied to use for the detection in the field and of various persistency pathogens.