Aberrant glycosylation is one of the major hallmarks of cancer with altered gene expression signatures of sialyltransferases. ST3Gal-I, a sialyltransferase, is known to play a crucial role in sialylation of T antigen in bladder cancer and it has reported elevated expression in breast carcinogenesis with increased tumor progression stages. The aim of the current study is to develop new monoclonal antibodies (mAbs) against human ST3Gal-I and evaluate their diagnostic potential. We developed a repertoire of stable hybridoma cell lines producing high-affinity IgG antibodies against recombinant human ST3Gal-I, expressed in E. coli BL21-DE3 strain. In order to demonstrate the diagnostic value of the mAbs, various clones were employed for the immunohistochemistry analysis of ST3Gal-I expression in cancerous tissues. Antibodies generated by 7E51C83A10 clone demonstrated a strong and specific fluorescence staining in breast cancer tissue sections and did not exhibit significant background in fibroadenoma sections. In conclusion, the mAbs raised against recombinant ST3Gal-I recognize cellular ST3Gal-I and represent a promising diagnostic tool for the immunodetection of ST3Gal-I expressing cells. Specific-reactivity of clone 7E51C83A10 mAbs towards ST3Gal-I was also confirmed by immunoblotting. Therefore, our observations warrant evaluation of ST3Gal-I as a potential marker for cancer diagnosis at larger scale.