Objective to investigate the clinical significance of samples except skeletal muscle that could be detected the large-scale single deletions directly in the diagnosis of Kearns-Sayre syndrome by concluding the clinical and genetic features of KSS. Methods The clinical data of four patients with KSS, which were diagnosed from 2016 to 2017 by Beijing Children's Hospital, Capital Medical University, were collected. The clinical features and gene mutation characteristics were analyzed retrospectively. Results 4 patients were all consistent with the diagnosis criteria of KSS, the age of onset was 8.2 years old on average (5.0-11.8), and the initial symptoms were no specificity. Molecular analysis of the four cases revealed that the large-scale single deletions of mitochondrial DNA from the peripheral blood, the urine and other samples (muscle tissue, etc.) through the next-generation sequencing. Two cases for homogeneous variation. Of three cases who did pedigree analysis, only the mom of case 4 was detected the same variation with the proband. Conclusions With the development of the next-generation sequencing, the diagnosis of Kearns-Sayre syndrome was no longer than depending on the muscle biopsy. It is indeed possible to detect the large-scale single deletions in peripheral blood, urine and other samples, to improve the molecular diagnosis of KSS, which will have a significant influence on the diagnosis and management of KSS, but more cases should be needed in the future.