‘Neonatal’ Nav1.5 (nNav1.5) is a potent tumour metastasis marker found especially in aggressive human breast cancer cells in vitro, in tumour tissues of in vivo metastatic animal models and in patients positive for lymph-node metastasis. Its expression has been recently described in human brain neuroblastoma and astrocytoma. However, a thorough understanding of nNav1.5’s role in cancers has been limited by the lack of specific antibodies against it. Here, a mouse monoclonal antibody, 4H8 mAb-nNav1.5, was obtained and characterised concerning its efficacy in detecting nNav1.5 using indirect ELISA, surface plasmon resonance (SPR), Western blotting and immunofluorescence microscopy. 4H8 mAb-nNav1.5 was selected from a panel of hybridoma clones raised against nNav1.5 specific peptide (15 mers). The antibody exhibited linear association against nNav1.5 specific-linear peptide in indirect ELISA and was supported by SPR. The antibody also demonstrated strong immunoreactivity in immunofluorescence imaging of nNav1.5-abundant cells, human and mouse aggressive breast cancer cells, MDA-MB-231 and 4T1, respectively, which was not observed in nNav1.5-deficient cells, human less aggressive breast cancer cells, MCF-7 and non-cancerous breast epithelial cells, MCF-10A. This study demonstrates the initial description of 4H8 mAb-nNav1.5, which could serve as a beneficial tool to enhance future studies on nNav1.5 expression and function in cancers.