RNA sequencing and short-read assembly was utilized to produce a transcriptome of ovarian tissues from three-year-old diploid and tetraploid loaches (Misgurnus anguillicaudatus). A total of 28,369 unigenes were obtained, comprising 10,546 unigenes with length longer than 1000 bp. More than 73% of the unigenes were annotated through sequence comparison with databases. The RNA-seq data revealed that 2253 genes were differentially expressed between diploid and tetraploid loaches, including 1263 up-regulated and 990 down-regulated genes in tetraploid loach. Some differentially expressed genes, such as vitellogenin (Vtg), gonadotropin releasing hormone receptor type A (GnRHRA), steroidogenic acute regulatory protein (StAR), mitogen-activated protein kinase 14a (MAPK14a), ATP synthase subunit alpha (atp5a), and synaptonemal complex protein 1 (Scp1), were involved in regulation of cell proliferation, division, gene transcription, ovarian development and energy metabolism, suggesting that these genes were related to egg diameter of the loach. Results of transcriptome profiling here were validated using real time quantitative PCR in ten selected genes. The present study provided insights into the transcriptome profile of ovarian tissues from diploid and tetraploid loaches Misgurnus anguillicaudatus, which was made available to the research community for functional genomics, comparative genomics, polyploidy evolution and molecular breeding of this loach and other related species.