Abstract: RNA interference, mediated by small interfering RNA, is a fast developed post-transcriptional gene silencing method in resent years, which has been extensively used in the fields of gene function research, gene networks regulation and disease therapy, etc. Most siRNA expression vectors dependent on one of the RNA polymerase III promoters, operating small hairpin RNA expressing in cell or in vivo. These promoters mainly include human and mouse U6 promoters and human H1 promoter. In this study, in order to explore whether fish RNA polymerase III promoters could efficiently drive shRNA expression in vivo, and consequently make better use of RNAi to research gene function and virus resistance in fish, three shRNA expression vectorspZH1siGCRV-CMVeGFP, pZU6siGCRV-CMVeGFP and pCH1siGCRV-CMVeGFP were constructed by employing zebrafish H1/U6 promoters and grass carp H1 promoter respectively, using outer capsid protein VP7 gene of grass carp reovirus (GCRV) as target gene and eGFP as report gene. Then the three vectors were injected into rare minnow embryos respectively. Since siRNA was short and difficult to be detected, stem-loop RT-PCR technique has been introduced to detect shRNA expression in different development stages of rare minnow embryos. Experiment results have showed that all of the three fish RNA polymerase III promoters could efficiently drive shRNA expression. At the same time, GCRV siRNA can be detected in all of the sampling embryo development stages. And stem-loop RT-PCR technique was proved to be a simple and facile tool to detect siRNA. The construction of efficient and lasting siRNA expressing vector in fish embryo and establishment of easy and fast siRNA detection method have provided a solid foundation and forceful technique support for producing GCRV resistant transgenic fish and studying siRNA interfering mechanisms in virus replication.