The myxozoan parasite Enteromyxum scophthalmi causes severe enteritis in cultured turbot Scophthalmus maximus, thus generating important economic losses. At present, there are no prevention or control measures for the disease, and many aspects of the life cycle and transmission of the parasite are not yet known. In this study, a highly sensitive, reproducible and rapid quantitative (real time) polymerase chain reaction (qPCR) assay was developed to detect E. scophthalmi DNA. The qPCR assay targets the 28S rRNA gene of the parasite, which has a high identity (94%) with the myxosporidian Enteromyxum leei rRNA gene. The qPCR assay was able to detect up to 13 DNA copies, corresponding to 0.55 fg, estimating that genomic DNA has around 1450 copies of 28S rRNA gene per parasite nucleus. The mean intra- and inter-assay coefficients of variation were below 5% and no detectable amplification was observed with DNA from non-infected turbot. The assay was validated with a histological identification of intestinal content samples from experimentally infected turbot and a good correlation between both methods was observed. The results demonstrate that the qPCR assay can be applied in the diagnosis of turbot enteromyxosis and to determine the relative abundance of E. scophthalmi in turbot intestinal contents in health monitoring studies.