We performed the metatranscriptomic analysis to explore the novel eukaryotic lignocellulolytic genes from enrichment culture with pretreated-rice straw as carbon source. The eukaryotic mRNA was sequenced on GS FLX Titanium system and assembled. From the result of analyzing of sequence datasets, 147 glycoside hydrolase (GH) genes were indentified and classified into 40 GH families, and almost genes showed less than 70% similarity to known enzymes of eukaryota including thermophilic fungi and alveolata. Furthermore, 14 contigs were predicted to be full-length cDNA. This study shows that metatranscriptomic analysis using next-generation sequencer is possible to not only to access the transcriptome information but also to obtain full-length cDNA sequences.