A reverse transcription-polymerase chain reaction (RT-PCR) was developed for detection of Rabbit haemorrhagic disease virus (RHDV). Three primers were designed according to the sequences published in GenBank and different combinations of primers were screened for the best results. The amplification conditions, including concentration of Mg2+and annealing temperature were optimized. The sensitivity and the specificity of RT-PCR developed in the present study were also tested. A target fragment of 331 bp was amplified using the selected primers. The optimal concentration of Mg2+was from 1.0 mmol/L to 1.5 mmol/L and annealing temperature in the range of 50℃-60℃did not show differences. The minimum detectable copy number of the template was 102. In addition, this method had no cross reaction with tested rabbit Rotavirus and Vesicular stomatitis virus. This method was also compared with commonly used HA test. The results showed that RT-PCR detected 3 more samples than HA test thus the positive rate increased from 66.7%to 77.8%. The RT-PCR developed here was a specific, sensitive and rapid diagnostic method, which could be used for clinical diagnosis and prevention and control of rabbit haemorrhagic disease.%根据GenBank公布的兔出血热病毒（Rabbit haemorrhagic disease virus，RHDV）序列，设计了3条引物，对引物组合进行了筛选，优化PCR体系各反应条件，测定该方法的敏感性与特异性，并进行临床应用对比试验。结果显示，筛选出的引物组合能够特异性扩增331 bp产物，Mg2+浓度1.0~1.5 mmol/L时扩增效果最好，退火温度在50℃~60℃之间扩增无差异性。该方法可以扩增102拷贝以上的模板，对兔轮状病毒与水泡性口炎病毒检测结果均为阴性；临床应用对比实验显示，与HA检测相比，该方法检出阳性率由66.7%提升至77.8%。本研究建立的RT-PCR诊断方法具有特异、灵敏、快速等特点，能够用于该病的临床检测，为防控该病提供了技术保障。
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