Transglutaminase (TGase) is an enzyme that catalyzes acyl transfer reactions by creating covalent cross-links between protein molecules and has been used to improve the physical and functional properties of protein-based foods. The objectives of this study were the extraction, purification, and biochemical characterization of TGase from sardine (Sardina pilchardus) flesh in order to provide a suitable TGase enzyme for food industry applications. The results showed a specific activity, yield, and purification fold of 357.14 U/mg protein, 36.74%, and 183.15, respectively. The enzyme exhibited maximal activity at 40 °C and pH 8.0, with a molecular weight of around 57 kDa. The effect of time on TGase thermal stability at 40 °C showed a gradual decrease in its catalytic activity during the incubation time until the enzyme was completely inactivated at 60 min. Additionally, the sardine TGase was found to be calcium-dependent. However, Mg²⁺ and Ba²⁺ ions were found to be effective in its activation to some extent and a total inhibition was shown by Zn²⁺ and Sr²⁺ ions. The TGase activity was affected markedly by NaCl and EDTA, and lost, respectively, about 80.7% and 36.49% from its activity by increasing the concentration (1.5 M NaCl and 20 mM EDTA). Based on the surface hydrophobicity and solubility results, the cross-linking of natural actomyosin mediated by TGase increased to a greater extent. The results revealed that sardine TGase possessed attractive qualities, making it a potential alternative to other TGase sources for food industry applications.