Red-spotted grouper nervous necrosis virus (RGNNV) is one major pathogen of aquaculture. It mainly affects the larvae and juveniles of groupers. RGNNV is composed of two single stranded RNA (RNA1 and RNA2). Ozone can inactivate RNA virus by degradation of viral genome. So treatment by ozone is a common measure to disinfect RGNNV in farms. But it has no efficient methods to evaluate the disinfection effect of ozone up to now. In this report, a special RT-PCR named as LR RT-PCR (Left reverse transcript Right amplify RT-PCR) was developed and optimized to evaluate degradation effects of RGNNV RNA2 by ozone. Based on the 3 end and 5 end of RGNNV RNA2, a special reverse transcription primer and a set of PCR primers were designed respectively. The concentration of primers, Mg2+, dNTPs as well as annealing temperature of LR RT-PCR was optimized in this study. The sensitivity and specificity of LR RT-PCR also were confirmed. Finally the LR RT-PCR method was used to evaluate degradation effects of RGNNV RNA2 by ozone. With summarizing the results of the experiment, in 25 μl of reaction volume, the optimized parameters of LR RT-PCR were primers 0.2 μmol/L, Mg2+ 4 mmol/L, dNTPs 0.5 nmol, Ex Taq 0.5 U and cDNA template 1 μl. The annealing temperature was 60℃. The sensitivity of LR RT-PCR was 1 pg to RGNNV RNA2 and there were no cross reactions with genomic RNA from healthy groupers, DNA from common bacteria and viruses of aquaculture. Comparative analysis of LR RT-PCR and routine RT-PCR was carried out to evaluate the destructive effects by ozone. As the concentration of ozone increased from 0.3 mg/L to 2 mg/L in solution of RGNNV RNA2, the amplified product of LR RT-PCR decreased and finally was undetected. Former reported routine RT-PCR method for RGNNV detection did not reflect above tendency. This report demonstrated that the LR RT-PCR can quickly and accurately evaluate disinfection effects of RGNNV by ozone and can be widely used in hatcheries of groupers.