Technology relates to an antibody against phosphorylated Ser-125-NPM1, its production method and use of highly specific antibodies against phosphorylated NPM1. NPM1 is identified as a novel substrate for Aurora kinases A and B, both acting on same site, Ser125 on NPM1 for phosphorylation during the onset of mitosis. Technology also discloses a method and kit used for production and usage of polyclonal antibodies specifically targeting Ser125 phosphorylated NPM1 along with pharmaceutically acceptable carrier could be used in diagnosis of tumor.