BackgroundAntimicrobial peptides are small, cationic, amphipathic peptides composed of 10–50 amino acids, playing a crucial role in innate immunity. While they are found in both vertebrates and invertebrates, they are particularly effective in invertebrates. This study presents the identification and molecular characterization of the potential antimicrobial peptide thymosin from Pearl spot, Etroplus suratensis (Es-Thyβ). Phylogenetic analysis and structural characterization of the identified peptide are analyzed and substantiated through the study. ResultsThe sequence analysis of Es-Thyβ revealed an Open Reading Frame (ORF) of 132 nucleotides, encoding a 43-amino acid peptide. The hydrophobicity of the peptide, determined using PepDraw, was found to be +68.93 Kcal*mol−1. Through in silico analysis the identified Thymosin was determined to be Es-Thyβ. The structure and characteristics of the peptide sequence were further analyzed using multiple in silico software tools, providing confirmation of its identity as Es-Thyβ. ConclusionThe study identified and described a potential antimicrobial peptide Es-Thyβ, a member of the thymosin β4 family, with conserved sequences and evolutionary links across teleost fishes.