Abstract: Objectives: Thyroid dysfunction such as hypothyroidism (THO) and hyperthyroidism (THE) are the disease caused by pathological processes in the thyroid. The current diagnosis of thyroid dysfunction is variable because of ages and genders. The aim of this study was to explore the novel candidate biomarker panels for hypothyroidism and hyperthyroidism screening with mass spectrometry and bioinformatics. Methods: Plasma samples were collected from 15 THE patients, 9 THO patients, and 15 healthy controls. DIA-based proteomic and untargeted metabolomic analyses were performed to identify the novel biomarker panels for THO and THE. Finally, three candidate biomarkers were verified by ELISA in 34 samples. Results: A total of 2738 proteins and 6103 metabolites were identified, and 173 proteins and 2487 metabolites were found to be differentially expressed among THE, THO and control groups. The results of the ensemble feature selection, K-means clustering and the least absolute shrinkage and selection operator (LASSO) regression model showed that four proteins (C4A, C3/C5 convertase, APOL1, and ITIH4) and four metabolites (L-arginine, L-proline, cortisol, and cortisone) identified by plasma proteomics and metabolomics could help distinguish THO and THE patients from healthy controls. Conclusions: This study identified and verified two pairs of biomarker panels that can distinguish the THE and THO patients regardless of ages and genders. Consequently, our findings represent a comprehensive analyses of thyroid dysfunction plasma, which is significant for the clinical diagnosis.