Background: Tanshinone IIA is effective in the treatment of various cardiovascular diseases. Currently, tanshinone IIA is primarily derived from the roots and rhizomes of Salviamiltiorrhiza Bunge. However, resources of S.miltiorrhiza are already facing scarcity. Objectives: The aim of this study was to obtain an alternative and high-yield mutagenic strain of tanshinone IIA, thus establishing a cost-effective and non-plant-derived substitute for tanshinone IIA. Methods: The mutants with enhanced tanshinone IIA production were selected through protoplast mutation induced by ultraviolet radiation (UV) and sodium nitrite (NaNO2). The yield of tanshinone IIA (YT) was determined using a TU-1810 ultraviolet-visible (UV/Vis) Spectrophotometer and LC-2010A high-performance liquid chromatography (HPLC). The selected mutant strains with the highest yield were subjected to continuous cultivation, and the dry weight of mycelium (DW) and the YT in the first generation were used as controls to assess passage stability. Fungal genomic DNA from the wild-type and mutant strains was extracted from axenic cultures using the modified cetyltrimethyl ammonium bromide (CTAB) method. Results: The colony morphological characteristics exhibited significant differences between the wild-type TR21 and the mutant NU204. The NU204 demonstrated a significantly higher tanshinone IIA yield at 165 ± 2.52 μg/g, which was 4.67-fold greater than that of TR21 (P < 0.01), indicating a substantial enhancement in NU204 production compared to TR21. The NU204 underwent continuous cultivation for five generations, and there were no significant differences in the DW and YT among the different generations (P > 0.05). The observed genetic changes between TR21 and NU204 were induced by the protoplast mutation. Conclusions: The mutant NU204 is emerging as a promising novel alternative source for tanshinone IIA, offering potential practical applications in production.