Emerging and existing diseases are the major havoc to the shrimp aquaculture industry. Outbreaks of viral epidemics severely hinder the sustainable farming system with significant economic losses worldwide. The Pacific white shrimp, Penaeus vannamei farming was seriously affected by infectious myonecrosis caused by a double-stranded RNA virus, infectious myonecrosis virus (IMNV). Early and rapid diagnostics is the priority on considering the efficient management and prevention measures. So, the present study utilized the accurate, rapid and specific detection capabilities of real-time PCR on SYBR Green platform to diagnose and quantify the viral load from the infected tissues by designing an efficient PCR primer set. The developed PCR could detect the virus at 98% efficiency with 10 viral copy number as the limit of detection. The standard curve analysis and amplification arithmetics have shown that it can even detect even less than 10 copy numbers of virus in a sample. The standard curve of the assay has shown R2 value of 0.98 and slope of −3.3834 without any significant variations in inter- and intra- assays. The validated PCR primer pairs and developed SYBR green-based real-time PCR is highly specific, equally sensitive and comparatively economic than the existing TaqMan probe-based PCR for detection of IMNV. •Validated a new set of PCR primers for SYBR green-based qPCR for the detection and quantification of IMNV infection in India•The developed PCR could detect the virus at 98% efficiency with 10 viral copy number as the limit of detection.•The standard curve of assay was showing an R2 value of 0.98 and a slope of −3.3834 without any statistical variations.•The assay can be implemented for the accurate diagnosis and viral load quantification of the IMNV from infected shrimps.