Reverse transcription (RT) of viral RNA to complementary DNA (cDNA) is essential for mycovirus detection; however, the stable secondary structures of double-stranded RNA (dsRNA) and the genomic diversity of viruses complicate this process. This study systematically optimized RT conditions for 15 mycoviruses infecting Lentinula edodes using RT-qPCR, focusing on thermal denaturation temperatures (no heat denaturation [CK], 65 °C [T65], 95 °C [T95], and 98 °C [T98]) and dimethyl sulfoxide (DMSO) concentrations (0–20%). For purified LeV-HKB dsRNA, T95 and T98 increased cDNA yields by over 50,000- and 100,000-fold, respectively, compared to T65. In total RNA extracts from infected L. edodes, T98 increased cDNA yields for dsRNA mycoviruses by approximately 5-fold compared to CK. Conversely, T95/T98 did not improve cDNA yields for ssRNA viruses, and even resulted in slightly reduced yields for certain viruses. DMSO exerted virus-specific and temperature-dependent effects on RT efficiency: moderate concentrations (5–15%) enhanced cDNA yields at CK or T65, whereas high concentrations (e.g., 20%) reduced yields at T95 or T98. No universal DMSO concentration was optimal across viruses, necessitating tailored approaches. Overall, T98 is recommended for dsRNA viruses, CK/T65 for ssRNA viruses, and T98 as a practical compromise for mixed RNA types. This protocol improves detection sensitivity and reliability, thereby facilitating diagnosis and research on fungal RNA viruses.