Austwickia chelonae infects reptiles, birds, mammals, etc., causing skin diseases or deaths in the wild and domestic animals. In recent years, this pathogen outbroke in the rescue population of the first-class protected animal—Crocodile Lizards (Shinisaurus crocodilurus). Traditional methods for pathogen detection are labor-consuming and time-costing. Therefore, this study developed a highly specific and convenient method for the detection of Austwickia chelonae based on its whole genome sequence, and applied this method to the risk prediction of skin disease in crocodile lizards. Among the three pairs of specific primers for A. chelonae detection developed in this paper, primer set AC3 shows the best results.