Many genetic disorders are complicated with variety of clinical signs and multiple causes, which preventing an accurate diagnosis and the understanding of genetic basis. Recent advances in next-generation sequencing (NGS) technologies make it possible to comprehensively explore and identify disease-associated variants and genetic variants that could be associated with disease susceptibility. Many patients with a suspected genetic disorder will present with similar features due to the clinical variability and the variable expressivity among this group of disorders. A total of 25 targeted NGS panels was developed for a variety of disorders such as epilepsy, neuromuscular disease, arrhythmia, metabolic disorder, dimorphism, autism disorder, hearing loss, skeletal dysplasia, familial cancer, and so on. For clinical diagnosis of 187 patients, the targeted NGS panels was performed instead of the serial single gene testing. Of the 187cases, a molecular diagnosis with the causative variants was provided for 58 cases (27.2%). And the potential diagnosis cases with variants identified in clinically relevant genes were made for 54 cases (28.9%) that needed the additional segregation analysis. In this study, we showed that targeted NGS panels has been successfully applied as a cost-effective diagnostic tool compare with a single- gene test for the first step of the genetic evaluation.