A recombinant bovine serum albumin mature peptide, having an amino acid residue sequence as shown in SEQ ID NO : 1, and an optimized nucleotide sequence encoding the recombinant bovine serum albumin mature peptide as shown in SEQ ID NO : 3. A preparation method for the recombinant bovine serum albumin mature peptide comprises : synthesizing an optimized gene of a bovine serum albumin mature peptide; then integrating the gene into an expression vector, and using the vector to transform various host cells for expression; culturing the transformed host cells to obtain a fermentation broth containing a large amount of a recombinant protein; and further separating and purifying the fermentation broth to give the product. Under mass spectrometry the above recombinant bovine serum albumin is completely consistent with a natural bovine serum albumin mature peptide sequence, has the advantage of being free from animal virus contamination and has high purity, is a natural bovine serum albumin substitute, and can be applied in biochemical pharmaceuticals and other industries.