The invention discloses a construction method of mycobacterium tuberculosis fusion protein Mtb 10.4-Hsp16.3, an expression method thereof, a purification method thereof and application thereof. The construction method comprises the following steps: performing the PCR (polymerase chain reaction) amplification to Mtb 10.4 gene and Hsp16.3 gene, and sequentially inserting the amplified genes into a multi-clone site of a cloning vector to construct a recombinant vector; then expressing in the colibacillus; and finally purifying to obtain the fusion protein Mtb 10.4-Hsp16.3, wherein the fusion protein can be applied in a tuberculosis subunit vaccine. The invention has the advantages that the fusion protein vaccine can induce the higher cell of the animal to react with body fluid immunoreaction; the animal challenge test shows that the vaccine plays a part in immunity enhancing on the basis of the BCG (bacille calmette guerin) immunity, so that the protective effect of the BCG can be improved and prolonged; and the vaccine has no obvious side effects.