The invention discloses a preparation method of helicobacter pylori serine protease. The preparation method specifically comprises the following steps: amplifying a nucleotide sequence shown in SEQ ID No. 2 by using SEQ ID No. 3 and SEQ ID No. 4, then performing enzyme digestion on an amplified product, connecting the amplified product into a pMAL-C plasmid which is subjected to same enzyme digestion to obtain a recombinant expression vector pMAL-C-SP, then performing induced expression on the recombinant expression vector pMAL-C-SP in escherichia coli, then purifying by using an Ni2<+> affinity chromatography column and a strong anion exchange column, and finally screening by using an SephadexG200 molecular sieve to obtain helicobacter pylori serine protease. Helicobacter pylori serine protease obtained by using the method disclosed by the invention is large in quantity and small in loss, has serine protease activity, can be used for laying a solid foundation for researching crystal structures and functions of protein, and can also be used as a research and development target spot of pancreatin substitutes and medicines with wide prospects.