Background <p>Protoplasts have been widely utilized in tissue culture, single-cell RNA sequencing, and transient gene expression analyses. However, the primary challenge in their preparation lies in the efficient removal of rigid cell walls to obtain high-quality plant cells. The aim of this study is to establish an effective method for protoplast isolation from saffron (<i>Crocus sativus</i> L.), thereby accelerating advances in molecular biology research in this species.</p> Result <p>Tissue-specific protoplasts were successfully isolated from apical buds, petals, leaves, and roots of saffron, with yields of approximately 8.81 × 10⁶, 1.36 × 10⁷, 7.84 × 10⁵, and 5.69 × 10⁵/g FW, and viabilities of ~ 95%, ~ 94%, ~ 96%, and ~ 61%, respectively. The optimal enzymatic digestion conditions varied by tissue type: apical buds (0.6&#xa0;M D-mannitol, 1.5% cellulase, 0.5% macerozyme, 28&#xa0;°C, 3&#xa0;h); roots (0.7&#xa0;M D-mannitol, 2.0% cellulase, 0.5% macerozyme, 0.5% pectinase, 28&#xa0;°C, 4&#xa0;h); petals (0.7&#xa0;M D-mannitol, 1.5% cellulase, 0.5% macerozyme, 28&#xa0;°C, 3.5&#xa0;h); and mesophyll (0.7&#xa0;M D-mannitol, 1.5% cellulase, 0.5% macerozyme, 28&#xa0;°C, 4&#xa0;h). The quality of the protoplasts was affected by developmental stages and sampling locations. Furthermore, two high-quality single-cell libraries were constructed using protoplasts from non-flowering and flowering apical buds, respectively. We successfully visualized the subcellular localization of GFP in the mesophyll protoplasts.</p> Conclusion <p>This study presents the first detailed report on protoplast isolation, evaluation, and application in saffron. By overcoming the technical challenges of tissue-specific protoplast isolation from diverse organs, we established a highly efficient method that enables both the development of novel germplasm and mechanistic insights into saffron organogenesis, growth, and development. </p> Graphical Abstract <p></p>