Carnitine transport and cycle disorders (CCD) are a group of metabolic disorders characterized by either carnitine depletion or dysfunction in the carnitine cycle, a critical process for the transport of fatty acids into the mitochondria and their subsequent β-oxidation. Clinically, CCD can manifest with a wide range of symptoms, including hypoketotic hypoglycemia, which may be accompanied by signs of liver dysfunction, hepatic steatosis, myopathy and cardiomyopathy. Biochemical diagnosis typically involves measuring carnitine and acylcarnitine levels in blood, alongside organic acid profiling in urine. However, due to phenotypic overlaps with other metabolic disorders, precise molecular diagnosis is essential for accurate disease classification and subtype determination. The present study aimed to develop and clinically validate a novel CCD panel, specifically designed for Oxford Nanopore Technologies (ONT) platform compatibility. The panel targeted four key CCD related genes (CPT-1, CPT-2, SLC22A5 and SLC25A20). An amplification-based library preparation method pooling 21 primers specific to the CCD-related genes into two tubes was optimized. The panel was then applied to screen 20 patients previously diagnosed with CCD via second-generation sequencing platform. Comparative analysis of results from both platforms revealed a 100% concordance in detecting pathogenic, likely pathogenic, and variants of unknown significance associated with CCD. In silico analysis was also performed to predict the pathogenic potential of the variants of unknown significance. Here we report the development and clinical validation of a multi-gene diagnostic panel for ONT platform. The results demonstrated the feasibility of ONT-based genetic testing for CCD and set the stage for the development of similar diagnostic panels for other genetic disorders, offering a streamlined and putatively cost-effective alternative to current sequencing methodologies.