The aim of this study is to determine serum anti-melanoma differentiation-associated gene 5 prevalence and their clinical associations in Chinese patients with polymyositis and dermatomyositis (PM/DM). Serum anti-MDA5 antibody was detected by ELISA in 113 adult PM/DM patients and in various controls. Flow cytometry was applied to analyze the subgroups of lymphocytes in the peripheral blood of PM/DM patients. The serum anti-MDA5-positive rate in the DM patients (22.6%) was significantly higher compared with that in PM patients (0%, P < 0.005), patients with SLE (3.3%, P < 0.05), RA (3.3%, P < 0.05), pSS (0%, P < 0.05), pulmonary infection (0%, P < 0.05) and healthy controls (0%, P < 0.001). The percentage of decreased CD4(+), CD8(+) T cell counts, raised CD4(+)/CD8(+) ratio in peripheral blood and the incidence of acute/subacute interstitial pneumonia (A/SIP) were significantly higher in anti-MDA5-positive DM group than negative group (all P < 0.05). Additionally, logistic multivariate analysis showed that anti-MDA5 is an independent risk factor for death of ILD in DM (OR = 8.46, 95% CI 1.77-40.36, P = 0.007). In conclusion, in Chinese PM/DM patients, serum anti-MDA5 antibody is mainly presented in DM patients and can be a useful marker for A/SIP in patients with DM. It can predict unfavorable prognosis in DM patients with ILD. Further studies are needed to identify how the abnormal T cells in peripheral blood participated in the generation of the anti-MDA5 antibody.