The invention discloses BrBP1 polypeptide in specific binding with breast cancer brain metastases cells, and further relates to a preparation method of the polypeptide. The method includes: by utilization of phage display technology, carrying out a plurality of rounds of whole-cell depletion screening, and screening out phage clones bound with a breast cancer brain metastases cell line; randomly picking out a plurality of the phage clones, and selecting strongly positive phage clones with a cell enzyme-linked immuno sorbent assay (ELISA) method; and after increasing the strongly positive phage clones, carrying out sequencing, and synthesizing the tested BrBP1 polypeptide in a manual mode. The polypeptide can be applied to preparation of medicine used for curing breast cancer brain metastases and kits used for early-stage diagnosis of the breast cancer brain metastases. The polypeptide can be synthesized with a manual method, is small in molecular weight, high in activity, strong in penetrating power, high in affinity and specificity and low in toxicity, has good tumor-targeting performance inside and outside a body, can also be internalized to enter cells, and is suitable for being used as vectors for targeted therapy.