Mycoparasites are an assemblage of biotrophic and necrotrophic fungi that occur on plant pathogenic fungal hosts. Biotrophic mycoparasites are often overlooked in transcriptomic-based biocontrol studies. Sphaerodes mycoparasitica ( S.m. ) is a specific biotrophic mycoparasite of plant pathogenic Fusarium graminearum (F.g.) , a devastating Fusarium head blight (FHB) disease in small-grain cereals. To understand the biotrophic mycoparasitism comprehensively, we performed Illumina RNA-Seq transcriptomic study on the fungus-fungus interaction in vitro. The aim is to identify the transcript-level mechanism related to the biotrophic S.m. mycoparasitism, particularly its ability to effectively control the F.g. 3-ADON chemotype. A shift in the transcriptomic profile of the mycoparasite was triggered in response to its interaction with F.g. during recognition (1.5 days) and colonization (3.5 days) steps. RNA-Seq analysis revealed ~ 30% of annotated transcripts with "function unknown". Further, 14 differentially expressed genes functionally linked to the biotrophic mycoparasitism were validated by quantitative real-time PCR (qPCR). The gene expression patterns of the filamentous haemagglutinin/adhesin/attachment factor as well as cell wall-degrading glucanases and chitinases were upregulated by host interaction. Besides, mycoparasitism-associated antioxidant resistance genes encoding ATP-binding cassette (ABC) transporter(s) and glutathione synthetase(s) were upregulated. However, the thioredoxin reductase was downregulated which infers that this antioxidant gene can be used as a resistance marker to assess S.m. antifungal and antimycotoxigenic activities. The interactive transcriptome of S. mycoparasitica provides new insights into specific mycoparasitism and will contribute to future research in controlling FHB.