Abstract: White spot syndrome virus (WSSV) is a highly lethal and contagious viral pathogen of farmed shrimp and it causes substantial economic losses to the shrimp farming industry worldwide. During the last decade, although considerable progress has been made in the molecular characterization of WSSV, there is no effective therapeutic method available to interfere with the unrestrained occurrence and spread of the disease caused by the virus. So the best way to prevent WSSV infection is through pre-screening of WSSV-free broodstock or larvae and routine surveillance. Therefore, our goal is to establish a simple, rapid and accurate diagnostic method for WSSV in field conditions. In this study, a real-time fluorescence loop mediated isothermal amplification (RealAmp) was developed for WSSV diagnosis by using a portable fluorescence reader named ESE-Quant Tube Scanner, and then the RealAmp method was compared with nested PCR, real-time PCR and other 4 established LAMP methods in their sensitivity. The RealAmp method had a detection limit of 105 dilution template of genomic DNA within 30min under isothermal condition at 63℃, which was the same as that of the real-time PCR, but higher than that of nested PCR and other 4 established LAMP methods. Furthermore, the approach had no signal response to other DNA of shrimp pathogens including infectious hypodermal and hematopoietic necrosis virus (IHHNV). Also, sensitivity assay using pMD18-T-VP28 plasmid which contained the target sequence of the RealAmp method showed that the minimum detection limit was 24 copies of the plasmid within 30min. Moreover, 66 clinical samples of Penaeus vannamei were tested and compared by using the RealAmp method, nested PCR, real-time PCR and other 4 established LAMP methods. The result showed that the positive rate was 7.57% using the RealAmp method, which higher than that of nested PCR and other 4 established LAMP methods, and the accuracy was 100% after sequencing check. Overall, these data revealed that the RealAmp method had an equivalent to the real-time PCR method in specificity and sensitivity for WSSV detection, and has great potential as a field usable molecular tool for routine diagnosis of WSSV.