Display omitted •A TaqMan probe real-time PCR assay for rapid detection of Enterocytozoon hepatopenaei is developed targeting the small subunit ribosomal DNA sequence.•The qPCR assay is highly specific and the detection limit was down to 40 copies.•The standard curve showed a good linearity in detecting of EHP SSU rDNA plasmid from 4 × 102 to 4 × 108 copies/reaction.•The assay has diagnostic sensitivity and specificity of both 100% in comparison to SYBR green I qPCR. A TaqMan probe and a pair of specific primers were selected from the small subunit ribosomal DNA (SSU rDNA) sequence of Enterocytozoon hepatopenaei (EHP); this real-time PCR assay was developed and optimized. It showed a good linearity in detecting standards of EHP SSU rDNA fragments from 4 × 102 to 4 × 108 copies/reaction using the established method. The detection limit of the qPCR method was as low as 4 × 101 copies per reaction, which was higher than the conventional PCR and SYBR Green I-based EHP qPCR reported. Using the qPCR assay, EHP was detected in four batches of slow-growing Penaeus vannamei specimens collected from Tianjin and Zhejiang Province in China was detected using qPCR. The results showed that all the hepatopancreas from the slow-growing P. vannamei specimens were detected as EHP-positive. EHP copies of hepatopancreas in some batches had a negative correlation with the body mass index (BMI) of shrimps; however, not all batches of specimens had this negative correlation between EHP copies of hepatopancreas and BMI. This qPCR technique is sensitive, specific and easy to perform (96 tests in <3 h), which provides technical support for the detection and prevention of EHP.