Flaviviruses pose a significant threat to public health worldwide. Recently, a novel flavivirus, duck Tembusu virus (TMUV), was identified as the causative agent of a serious duck viral disease in Asia. Its rapid spread and expanded host range have raised substantial concerns regarding its potential threat to non-avian hosts, including humans. However, the specific molecular host responses to this virus are poorly understood. In this study, we used the RNA-sequencing technique to analyse the differential gene expression in the spleens of infected goslings 5days post-infection. In total, 2878 upregulated unigenes and 2943 downregulated unigenes were identified. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that different pattern recognition receptor (PRR) signalling pathways simultaneously participated in the sensing of the pathogen-associated molecular patterns (PAMPs) of TMUV, and the antigen presentation pathway and acquired immunity were activated. Then, the signals were transduced by the NF-kappa B (NF-κB) or the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways, resulting in the enormous production of various cytokines and interferon-stimulated genes (ISGs). We further investigated the immune response patterns in the liver and brain tissue using RT-qPCR. The bacterial peptidoglycan sensor nucleotide-binding oligomerization domain-containing protein 1 (NOD1) receptor was significantly upregulated, especially in the brain tissue, suggesting that NOD1 likely induces an inflammatory response by interacting with dsRNA, which is similar to its actions during hepatitis C viral (HCV) infection. However, major histocompatibility complex II (MHCII) was downregulated only in the spleen, indicating that the downregulation of MHCII in the spleen may be an immune evasion strategy of TMUV to facilitate pathogenesis during infection. Here, we are the first to report a transcriptome analysis of the host immune response to TMUV infection, and the data reported herein may help elucidate the molecular mechanisms of the gosling-TMUV interaction.