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We identified two tripartite motif (TRIM) genes, LcTRIM21and LcTRIM39, from the Asian Seabass Lates calcarifer, and examined their responses to experimental betanodavirus infection and stimulation with microbial pathogen‐associated molecular patterns. Genes encoding LcTRIM21and LcTRIM39were identified, cloned, and sequenced from the Asian Seabass. We analyzed the sequence using a variety of bioinformatics tools to determine protein structure, localization, and establish a phylogenetic tree. By using quantitative real‐time PCR, we analyzed expression profiles of the LcTRIM21and LcTRIM39genes in response to betanodavirus challenge as well as molecular pathogen‐associated molecular patterns like poly(I:C) and Zymosan A. The tissue distribution pattern of these genes was also examined in healthy animals. Asian Seabass homologues of the TRIMgene, LcTRIM21and LcTRIM39, were cloned, both encoding proteins with 547 amino acids. LcTRIM21is predicted to have an isoelectric point of 6.32 and a molecular mass of 62.11 kilodaltons, while LcTRIM39has an isoelectric point of 5.57 and a molecular mass of 62.11 kilodaltons. LcTRIM21and LcTRIM39homologues were predicted to be localized in cytoplasm by in silico protein localization. Structurally, both proteins contain an N‐terminal really interesting new gene (RING) zinc‐finger domain, B‐box domain, coiled‐coil domain and C‐terminal PRY/SPRY domain. Most tissues and organs examined showed constitutive expression of LcTRIM21and LcTRIM39. Upon poly(I:C) challenge or red‐spotted grouper nervous necrosis virus infection, LcTRIM21and LcTRIM39mRNA expression was significantly upregulated, suggesting that they may play a critical antiviral role against fish viruses. LcTRIM21and LcTRIM39expression were also upregulated by administration of the glucan Zymosan A. The TRIM‐containing gene is an E3 ubiquitin ligase that exhibits antiviral activity by targeting viral proteins via proteasome‐mediated ubiquitination. TRIMproteins can be explored for the discovery of antivirals and strategies to combat diseases like viral nervous necrosis, that threaten seabass aquaculture. Impact statementThis study explored the identification, cloning, and characterization of two TRIMgenes from Asian Seabass, an economically important fish, and their response to the viral pathogen betanodavirus that causes viral nervous necrosis disease in aquaculture. The findings of this study may help to develop species‐specific antivirals to reduce the risk of aquaculture infections caused by nodaviruses. This study explored the identification, cloning, and characterization of two TRIMgenes from Asian Seabass, an economically important fish, and their response to the viral pathogen betanodavirus that causes viral nervous necrosis disease in aquaculture. The findings of this study may help to develop species‐specific antivirals to reduce the risk of aquaculture infections caused by nodaviruses.