Abstract: Kidney disease has high incidence, which has become a major disease affecting people health nationwide. The discovery of new biological diagnostic markers is of great significance for early diagnosis and treatment of kidney disease patients. Mass spectrometry (MS)-based proteomics and peptidomics have become essential techniques in life science and clinical medicine, and have been powerful tools for early diagnosis, molecular typing, drug efficacy prediction, and prognosis assessment of diseases. Given that human plasma proteomics and peptidomics are complementary, in this study, the sequential precipitation and delipidation (SPD) method developed in our laboratory was used for the separation and enrichment of peptides in serum. Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which has fast, high sensitivity, and high-throughput characteristics, was used for the analysis of the peptides from serum of renal dialysis patients before and after dialysis, and then compared with healthy controls. Liquid chromatography-mass spectrometry (LC-MS/MS) was applied to analyze the peptidome of serum from renal dialysis patients and healthy controls. Eight and seven specific peptides are identified in the serum of renal dialysis patients and healthy controls, respectively. And compared with the serum of healthy controls, eight differentially expressed peptides in the serum of renal dialysis patients are also identified. The specific peptides in the serum of patients derive from fibrinogen chain α, histone H2B-1, α-HS-glycoprotein and coagulation factor XIII chain A, and the differentially expressed peptides mainly derive from fibrinogen chain α and coagulation factor XIII chain A. This study provides basic data for further validation of potential peptide biomarkers related to renal dialysis patients.