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<p>Abstract</p> <p>Background</p> <p><it>Clonorchis sinensis </it>is a zoonotic parasite causing clonorchiasis-associated human disease such as biliary calculi, cholecystitis, liver cirrhosis, and it is currently classified as carcinogenic to humans for cholangiocarcinoma. MicroRNAs (miRNAs) are non-coding, regulating small RNA molecules which are essential for the complex life cycles of parasites and are involved in parasitic infections. To identify and characterize miRNAs expressed in adult <it>C. sinensis </it>residing chronically in the biliary tract, we developed an integrative approach combining deep sequencing and bioinformatic predictions with stem-loop real-time PCR analysis.</p> <p>Results</p> <p>Here we report the use of this approach to identify and clone 6 new and 62,512 conserved <it>C. sinensis </it>miRNAs which belonged to 284 families. There was strong bias on families, family members and sequence nucleotides in <it>C. sinensis</it>. Uracil was the dominant nucleotide, particularly at positions 1, 14 and 22, which were located approximately at the beginning, middle and end of conserved miRNAs. There was no significant "seed region" at the first and ninth positions which were commonly found in human, animals and plants. Categorization of conserved miRNAs indicated that miRNAs of <it>C. sinensis </it>were still innovated and concentrated along three branches of the phylogenetic tree leading to bilaterians, insects and coelomates. There were two miRNA strategies in <it>C. sinensis </it>for its parasitic life: keeping a large category of miRNA families of different animals and keeping stringent conserved seed regions with high active innovation in other places of miRNAs mainly in the middle and the end, which were perfect for the parasite to perform its complex life style and for host changes.</p> <p>Conclusions</p> <p>The present study represented the first large scale characterization of <it>C. sinensis </it>miRNAs, which have implications for understanding the complex biology of this zoonotic parasite, as well as miRNA studies of other related species such as <it>Opisthorchis viverrini </it>and <it>Opisthorchis felineus </it>of human and animal health significance.</p>