The present disclosure provides an anti-biotin antibody, and provides an amino acid sequence encoding the CDRs of the antibody. Studies have shown that the antibody only reacts with a biotin conjugate or derivative, and does not react with free biotin. The present disclosure further provides applications of the antibody in, including but not limited to, ELISA, cell capture, sorting and enrichment, western blotting, flow cytometry, immunocytofluorescent staining, and immunohistochemistry. The anti-biotin antibody conjugated immunomagnetic beads can specifically and directly recognize a biotin labeled antigen, and do not bind to free biotin which is often presented in clinical samples and culture media. In addition, the anti-biotin antibody-conjugated magnetic beads or anti-biotin antibody-fluorescein provide an ideal solution for the isolation of specific cells, and can even enrich and separate target cells from samples rich in debris or other rare biological materials.