Building on this foundation, we successfully cloned and expressed the first luciferase from Antarctic krill (LAK). LAK emits bright violet fluorescence with a weak tissue penetration ability and is less likely to be absorbed by skin tissue, making it particularly suitable for shallow tissue imaging. At 25 \({}^{\circ}\)C, LAK exhibits high catalytic activity and sensitivity, with _Km_ values of 121 umol/L for ATP and 53.6 umol/L for the substrate luciferin, respectively. The minimum detectable level for ATP is as low as 1 fmol. However, the practical application of LAK is currently limited by its low thermal stability;after being heated at 50 \({}^{\circ}\)C for 10 min, the enzymatic activity of LAK is almost completely lost. Therefore, our objective was to engineer a LAK variant with enhanced thermostability and improved activity, characterized by a lower _Km_ and a higher _kcat_ value. Enhancing enzyme thermostability through protein engineering to withstand extremely harsh conditions is a significant objective in contemporary research. Enzyme thermostability is influenced by several key factors, including hydrogen bonds, strong covalent disulfide bonds, \(\pi\)-\(\pi\) interactions, and salt bridges. These structural features collectively contribute to the overall stability of proteins under high-temperature conditions. To achieve this goal, a variety of protein engineering techniques have been developed and refined. These techniques include random mutagenesis, which introduces random mutations to explore beneficial changes in protein structure; DNA shuffling, which mimics natural recombination processes to generate diverse variants; truncation, which involves the removal of specific regions of the protein to enhance stability; and circularization, which can stabilize proteins by altering their structural topology [18, 20, 21, 22, 23]. Each of these methods has demonstrated potential in improving the thermostability of enzymes, thereby expanding their application scope in industrial and biotechnological processes. The introduction of novel disulfide bonds into proteins has been a widely employed strategy to improve protein stability [20, 24, 25, 26]. However, not all engineered disulfide bonds result in increased stability, as there are instances where they have been reported to destabilize proteins. This highlights the need for more targeted approaches to reduce the screening effort required to identify enzymes with desired properties. Rational design based on protein structure has emerged as an efficient method to address this challenge [20, 27]. The structural diversity among enzymes complicates the relationship between enzyme structure and function, necessitating a detailed study of the characteristics of individual enzymes. LAK, in particular, differs significantly from traditional enzyme molecules in that it possesses multiple catalytic domains, including binding sites for the substrate (luciferin), ATP/AMP, and Mg\({}^{2+}\). Selecting appropriate mutation sites in such a complex enzyme is a formidable task. In this study, we introduced artificial disulfide bonds into LAK in the N-terminal and C-terminal connection region (linker peptide), the flexible surface loop region, and the C-terminal unstructured catalytic domain. We then investigated the effects of these mutations on the thermal stability and catalytic activity of LAK.