Scale drop diseases virus (SDDV), a newly characterized virus of farmed Asian sea bass (Lates calcarifer), has been reported in several countries in Southeast Asia. However, no fully validated detection method is publicly available for disease diagnosis and surveillance. Here, we described a newly developed semi-nested PCR (snPCR) method for detection of the virus from field samples. The designed primers targeting a gene encoding ATPase generated amplicons of 738 bp and 412 bp in the first and second step PCR, respectively. The established protocol could detect down to 100 viral copies/μL template and was 100-fold more sensitive than single step PCR. A Specificity test against extracted DNA from ten bacterial pathogens, tissues from viral infected specimens and fish host revealed no cross amplification. The SDDV snPCR method could detect the virus from all clinical samples showing symptoms of scale drop disease (n = 25) and all samples from outbreaks of an unknown disease (n = 6) whereas all clinically healthy fish sea bass (n = 161) and grouper (n = 45) collected from different provinces tested negative. The newly established protocol might be useful for Asian sea bass farming countries to initiate disease diagnosis and surveillance.