A nib gene of Soybean mosaic virus（SMV） strain 3 in northeastern regions of China was cloned from the SMV-infected soybean leaves. Sequence analysis of the nib gene exhibited the highest of 99.48% similarity with that from Ar33, followed by WS160, G4, G2, and L with 98.97%, 94.88%, 90.95% and 90.35%, respectively. The gene sequence also showed a distant phylogenetic relationship with that from 6202-2 and 6067-1 with 83.82% and84% of similarity respectively. The recombinant NIb protein was expressed in E.coliand purified using affinity chromatograph. Four monoclonal antibodies against NIb protein, 2G3, 1D6, 6F9 and 6B2 were prepared, by immunizing mice with the purified recombinant NIb protein. The results might lay the foundation for the function investigation of the SMV nib gene and soybean SMV resistance breeding.