Abstract: Ranavirus has a wide range of hosts, can infect reptiles, fish and amphibians, and lead to a high mortality. In order to further enrich its detection methods, in this study, a pair of specific primers was designed for the frog virus type 3 (FV3) based on its major capsid protein gene, and two other frog virus PCR detection methods were selected for comparison experiments. After the optimization of PCR reaction system, specificity and sensitivity test, a new PCR method for the detection of FV3 strain was established. This method has a minimum detection limit of 1.2 copies, has no cross-reaction with nervous necrosis virus, shrimp hemocyte iridovirus, largemouth bass Ranavirus, koi herpesvirus, carp edema virus, infectious spleen and kidney necrosis virus and Micropterus salmoides rhabdovirus. The present new method was used to detect the clinical samples, obtaining consistent results with the other two methods. The FV3 PCR detection method established in this research has the characteristics of simplicity, rapidity, good sensitivity, high specificity, and low cost. It can be used for rapid diagnosis and molecular epidemiological investigation of FV3 frog virus.