As one of the most hazardous jellyfish species in East Asian waters, Nemopilema nomurai is considered a key toxic component that triggers systemic toxicity reactions due to the high abundance of phospholipase A₂ (PLA₂) isoenzymes in its toxins. This study is based on a transcriptome database and identified four novel venom proteins with typical PLA₂ catalytic domains through sequence alignment and three-dimensional structural simulation. By constructing heterologous expression systems of pET-22b and pET-32a, it was found that the full-length recombinant protein presented as inclusion bodies in the E. coli BL21 (DE3) system. To obtain soluble phospholipase A₂, we innovatively adopted a multi domain truncation strategy and successfully constructed a truncated PLA₂-3 soluble expression system (pET-22b-PLA₂-3/BL21 (DE3)). In vitro activity testing confirmed its dose-dependent phospholipid hydrolysis activity. This work establishes for the first time a soluble heterologous expression system of Nemopilema nomurai PLA₂ toxin, laying the foundation for further elucidating its toxic molecular mechanism and developing specific detoxifiers.