Abstract: In view of the serious occurrence of root rot caused by Aphanomyces iridis in tobacco seedling bed of Henan Province in recent years, this study was carried out to clarify the biological characteristics of the pathogen and fro rapid detection at early stages. The biological characteristics of the pathogen were studied with the mycelial growth rate method, and specific amplification primers for A. iridis were designed and screened for application based on the CDS sequences of five coding genes of the representative strain CBS 524.87 in NCBI database. The results showed that the suitable temperature for mycelium growth of A. iridis was 25-35℃ on PDA plates, and the lethal temperature was 50℃ for 10 min. The suitable pH for A. iridis growth was 4.0-8.0 and the optimum pH was 6.0. Continuous light was beneficial to mycelium growth for A. iridis. Seven pairs of primers were designed and screened for the specific amplification of A. iridis, and the sensitivity to genomic DNA amplification was about 0.182 ng/μL. A. iridis could be specifically detected by the specific primer pair AiT3 for molecular detection of inoculated seedling substrate and tobacco seedlings respectively, with the detection sensitivity being 2.5×10^-2 g hyphae of per gram of substrate and 0.5 ng/μL tobacco seedling root genomic DNA. The results of this study provide technical support for the rapid molecular detection of A. iridis, and provide an important basis for the accurate identification and prediction of root rot caused by A. iridis at seedling stages.