Aim: The prevalence and diagnostic values of myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (MAAs) in dermatomyositis/polymyositis (DM/PM) were studied. Method: A commercial immunoblot assay with 16 autoantigens was used to detect MSAs and MAAs in serum samples from 130 DM/PM patients, 100 disease controls, and 50 healthy subjects. Results: The prevalence of anti-Jo-1, anti-MDA5, anti-TIF1 gamma, anti-Mi-2 beta, and anti-Mi-2 beta was significantly higher in DM/PM than in other connective-tissue diseases (CTDs). Moreover, anti-MDA5 and anti-Ro-52 were significantly higher in DM/PM with interstitial lung disease (ILD) than in DM/PM without ILD, while that of antiTIFly and anti-NXP2 were significantly lower in DM/PM with ILD than in DM/PM without ILD. For distinguishing DM/PM from other CTDs, the sensitivity, specificity, and positive predictive value (PPV) for anti-MDA5 were 28.46, 99.00, and 97.37%, respectively, with a positive likelihood ratio (LR +) of 28.46; they were 46.15, 58.00, and 58.82%, respectively, for anti-Ro-52 with an LR + of 1.10. For distinguishing DM/PM with ILD from DM/PM without ILD, the sensitivity, specificity, and PPV for anti-MDA5 were 45.57, 100.00, and 100.00%, respectively, and for anti-Ro-52 were 60.76, 73.91, and 80.00%, respectively. Conclusion: MSAs and MAAs serve as biomarkers for differentiating DM/PM from other CTDs as well as distinguishing DM/PM with ILD from DM/PM without ILD.