<p>Jellyfish play crucial roles in marine food webs, and accurately understanding their trophic dynamics is essential. Stable isotope analysis (SIA) has emerged as a valuable tool for unravelling these complexities. However, establishing clear protocols for jellyfish sample preparation is vital to avoid potential biases in isotopic data interpretation. In this study, we focused on <i>Nemopilema nomurai</i>, <i>Cyanea</i> spp. and <i>Aequorea</i> spp. medusae collected in the East Asian water, and measured their SI δ¹³C and δ¹⁵N values. Our results highlighted two principal sources that may influence the determination and interpretation of SIA for jellyfish: tissue-specific variation in SI compositions and the lipid effect on δ¹³C. Our data indicated significant tissue-specific differences in isotopic compositions, which could introduce substantial bias in SIA. The findings highlight the necessity of clearly removing jellyfish tentacles during tissue sampling for SIA, as their inclusion may lead to an overestimation of δ¹⁵N values. Furthermore, our results demonstrated a significant increase in both δ¹³C and δ¹⁵N values after lipid-extraction, emphasizing the importance of lipid correction for SIA. Notably, the δ¹³C values for three jellyfish after chemical lipid removal differed distinctly from the values predicted by generalized and species-specific (<i>Aurelia</i>) lipid corrections reported in previous studies. We thus highlight jellyfish species-specific of mathematical lipid normalization, which might due to the heterogeneity in lipid content among jellyfish species as proved by the variation in C:N ratios. Based on our findings, we proposed species-specific lipid corrected equations for <i>N. nomurai</i>, <i>Cyanea</i> spp. and <i>Aequorea</i> spp. Given the importance of accurately determining jellyfish trophic ecology in marine ecosystems, this study provides a refined methodology aimed at enhancing the precision of SIA for the jellyfish populations in the ecosystems of East Asian waters.</p>