The etiology of Keshan disease (KD), an endemic myocardiopathy in regions of China, is largely unknown. To show the protein changes in serum from KD patients versus controls and idiopathic dilated cardiomyopathy (IDCM) and to search specific biological markers for differential diagnosis for KD. Serum of 65 patients with KD was compared with 29 patients with IDCM, 62 controls from KD areas and 28 controls from non-KD areas by ClinProt/MALDI-ToF technique. The genetic algorithm, quick classifier algorithm and supervised neural network algorithm methods were used to screen marker proteins and establish diagnostic model. Thirty-four differential peaks were identified in KD patients compared with the healthy controls from non-KD areas. Thirty-eight differentially peaks were identified in KD patients and controls from KD areas; and sixty-seven differentially peaks were identified in patients with KD and patients with IDCM. We believe that marker protein peaks screened in KD patients, healthy controls and IDCM patients may provide clues for the differential diagnosis and treatment of KD.