AbstractKoi herpesvirus disease (KHVD) caused by the koi herpesvirus (KHV) is difficult to diagnose in live fish, presenting a challenge to the koi industry. The enzyme‐linked immunosorbent assay (ELISA) method cannot be widely used to detect KHV because few commercial anti‐KHV antibody exists. Here, we developed an anti‐ORF132 polyclonal antibody and confirmed its reactivity via indirect immunofluorescence assay and Western blotting. A double‐antibody sandwich ELISA (DAS‐ELISA) was established to detect KHV, monoclonal antibody 1B71B4 against ORF92 was used as the capture antibody, and the detection antibody was the polyclonal antibody against the truncated ORF132. The lowest limit was 1.56 ng/ml KHV. Furthermore, the DAS‐ELISA reacted with KHV isolates, while no cross‐reactions occurred with carp oedema virus, spring viraemia of carp virus, frog virus 3 and grass carp reovirus. Two hundred koi serum samples from Guangdong, China, were used in the DAS‐ELISA test, and the positive rate of the koi sera was 13%. The clinical sensitivity and specificity of the DAS‐ELISA relative to the traditional PCR method were 66.7% and 97.6%, respectively. Our findings may be useful for diagnosing and preventing KHVD in koi and common carp.