AbstractA nitrocellulose‐enzyme immunoassay (NC‐EIA) employing horseradish peroxidase (HRP) was developed for the detection of yellow‐head virus in the tissues of infected penaeid shrimp. Two substrates, DAB (3,3¢‐diaminobenzidine retrahydrochloride) and TMB (3,3¢5,5¢‐tetramethylbenzidine) for HRP were evaluated. The TMB was found to produce a more distinct and easily recognizable colour product and provided enhanced sensitivity (0.4 ng for TMB versus 0.8 ng viral protein for DAB). Under the present format, the NC‐EIA was capable of detecting as little as 100 TCID50of virus in infected tissues. Uninfected tissue homogenates yielded no significant backgrou