Koi herpes virus (KHV) has been recognized as a worldwide cause of mortality among populations of koi Cyprinus carpio koi and common carp. KHV disease can be diagnosed by the isolation of the virus, by anatomical assessment, and by the polymerase chain reaction (PCR) assay with agarose gel electrophoresis. Among these methods, the PCR assay has advantages over other methods in accuracy, but it retains a disadvantage with respect to quantification of the KHV genome. The TaqMan real-time PCR assay (TaqMan PCR assay) using the TaqMan-Probe has recently become more commonly used for the quantification of the KHV genome. However, it remains difficult to determine whether or not an amplified sequence originated from the KHV genome, because the TaqMan-Probe is degraded after PCR amplification. To overcome this problem, we utilized a novel quenching probe (QProbe) for quantitative PCR. This probe can be used to perform a melting curve analysis, thus eliminating the likelihood of obtaining false positives for the amplified product after PCR amplification. Two QProbes were designed to target SphI-5 and thymidine kinase, and these probes successfully detected the KHV genome; the amplified sequences could thus be evaluated by melting curve analysis.