Myostatin plays negative roles in muscle development. To block the inhibitory effects of myostatin on myogenesis, a 759 bp single chain variable fragment antibody (scFv) against myostatin was constructed and expressed in Escherichia coli. ELISA detection showed that the scFv could bind to myostatin, and change of the scFv N-terminal peptides decreased its binding affinity. MTT assay and cell morphology demonstrated that the cell number and viability of the C2C12 myoblast were enhanced by the scFv. Meanwhile, the scFv significantly inhibited the myostatin-induced expression of cyclin-dependent kinase inhibitor p21 and Smad binding element-luciferase activity. H2O2 increased the expression of Muscle RING Finger 1 (MuRF1) and Muscle Atrophy F-box (MAFbx) in myoblasts as well as myostatin and MuRF1 in myotubes, and the scFv significantly decreased the H2O2-elevated expression of these genes. Conclusively, the scFv we developed could antagonize the inhibitory effects of myostatin on myogenesis through Smad pathway and regulation of p21, MuRF1 and MAFbx gene expression. The scFv may have application in the therapy of muscular dystrophy and improvement of animal meat production.