The invention discloses a preparation method of a human active granzyme A recombinant protein. PCR amplification is carried out to obtain a human active granzyme A gene, the human active granzyme A gene is inserted to corresponding enzyme sites of a prokaryotic expression vector pET24a(+) to construct a recombinant expression plasmid pET24a(+)-aGzmA, and the pET24a(+)-aGzmA is transferred to Escherichia coli BL21 to obtain an engineering bacterium pET24a(+)-aGzmA/BL21. The engineering bacterium induced by IPTG can express the human active granzyme A recombinant protein, and the recombinant protein is expressed in an inclusion body form. The recombinant protein having a highest purity reaching above 95% can be obtained by separating through using a nickel affinity chromatography column, and an activity determination result of the recombinant protein by a BLT substrate solution shows that the recombinant protein has a good enzymatic hydrolysis activity. The method has the characteristics of low cost, simple operation, high protein expression level and the like, and the prepared recombinant protein has a biological activity and can be easily purified.