The nptⅡ gene, encoding neomycin phosphotransferase Ⅱ, is one of the most commonly used selection marker in transgenic research. In this study, recombinant NPTⅡ protein was expressed in E. coli and monoclonal antibodies were generated. The inheritance, temp-spatial profiling, abundance and subcellular localization properties were investigated using established Western blot (WB) analysis. The results showed that NPTⅡ protein in 0.25% of a single rice grain (about 0.025mg) was detectable, the expression of NPTⅡ protein is consistent with dominant inheritance law, positive seeds and homozygous lines can be identified in transgenic T₁ generation, furthermore, the abundance of NPTⅡ protein can provide clues for the identification of homozygous individual in T₁ seedlings. Quantitative analyses shows that NPTⅡ protein accounts for about 0.08‰ of total protein in the leaves at seedling stage. The expression profile of NPTⅡ protein in transgenic rice shows that the abundance of NPTⅡ driven by CaMV-35S promoter in the leaves and roots at seedling and adult stage are higher than that at tillering and booting stage. NPTⅡ was detectable at different stages in rice root, stem, leaf, panicle, flower and seed, however, the abundance of NPTⅡ in tillers, node, panicle axis and anther tissues is lower than others. In addition, the NPTⅡ protein is mainly localized in the soluble portion of cytoplasm. Taken together, an applicable immunoblot method was established and the expression properties of NPTⅡ protein in transgenic rice were demonstrated.