Suppressor tRNAs are engineered or naturally occurring transfer RNA molecules that have shown promise in gene therapy for diseases caused by nonsense mutations, which result in premature termination codons (PTCs) in coding sequence, leading to truncated, often nonfunctional proteins. Suppressor tRNAs can recognize and pair with these PTCs, allowing the ribosome to continue translation and produce a full-length protein. This review introduces the mechanism and development of suppressor tRNAs, compares suppressor tRNAs with other readthrough therapies, discusses their potential for clinical therapy, limitations, and obstacles. We also summarize the applications of suppressor tRNAs in both in vitro and in vivo, offering new insights into the research and treatment of nonsense mutation diseases.