Chronic progressive external ophthalmoplegia is a phenotype of mitochondrial disease and is mainly characterized by ptosis or eyeball movement disorder. This article reports the clinical and genetic analyses of a family with mitochondrial disease due to TWNK gene mutation. The proband was a woman aged 32 years， and she attended Department of Neuromuscular Disease， The Third Hospital of Hebei Medical University， due to blepharoptosis of both eyes for more than 10 years， with the manifestations of blepharoptosis and mild external ophthalmoplegia. She was diagnosed with chronic progressive external ophthalmoplegia， and similar cases were observed in her family. High-throughput sequencing and pathogenic mutation screening were performed for the proband to identify the pathogenic gene， and based on the clinical phenotype and sequencing results， the heterozygous mutation site （c.1411T>G， p.Y417D） of the TWNK gene was identified as a candidate pathogenic gene. Furthermore， Sanger sequencing was used for the validation of the proband and her family members， and the results confirmed the existence of this mutation site. According to the American College of Medical Genetics and Genomics guidelines， this mutation was determined as a pathogenic mutation （PS1+PM2+PP3）， with no previous reports in China. This study further expands the spectrum of TWNK gene mutations and provides a reference for the genetic counseling and molecular diagnosis of families with mitochondrial disease.