T7 endonucleaseⅠ(T7EⅠ) is a multiple functional endonuclease from bacteriophage T7, which can act as a reslovase to bind and resolve recombination intermediate Holliday Junction (HJ) specifically, act as a nicking enzyme to nick double-stranded DNA randomly, act as a specific endonuclease to recognize and cleave nicked sites and single-base mismatched sites on duplex DNA. Vector pET21a-MBP-T7EⅠ(E20K) and pET28a-T7EⅠ(E65K) were constructed, and MBP and His6 tagged T7EⅠ mutants MBP-T7EⅠ(E20K) and His6-T7EⅠ(E65K) were expressed and purified respectively. Two T7EⅠ mutants exist as homodimers, two domains of each mutant are inactive. One MBP-T7EⅠ(E20K) subunit and one His6-T7EⅠ(E65K) subunit can form a heterodimer, which have one active domain and one inactive domain (so named T7EⅠ-SAD). T7EⅠ-SAD was obtained by two approaches: The first concerns co-expression of two plasmids in E. coli strains ER2566 and purification of T7EⅠ- SAD by amylose and Ni-NTA affinity columns. The second concerns co-denaturing and co-refolding of MBP-T7EⅠ(E20K) and His6-T7EⅠ(E65K) in equal molar and isolation of heterodimer T7EⅠ-SAD by double affinity tagging. Using the cruciform structure-containing plasmid pUC(AT) and general closed circular plasmid pUC19 as substrates, the resolving, random nicking and nicked sites cleavage activity of T7EⅠ-SAD were analyzed. T7EⅠ- SAD can recognize cruciform structure, but it loses the capability to introduce two nicked sites simultaneously. In buffer with Mg²⁺, the specific nicking activity of T7EⅠ-SAD to pUC(AT) and random nicking activity to pUC19 are close to that of wide-type enzyme. In buffer with Mn²⁺, the random nicking activity of T7EⅠ-SAD is higher than wide-type T7EⅠ significantly. Results of stepwise denaturing and elution experiment showed that the subunits of T7EⅠ-SAD can disassociate in 5～6 mol/L urea or 1.75～2.0 mol/L guanidine hydrochloride. Gel-shift assay showed T7EⅠ-SAD can binding to four-way junction substrate Junction3 specifically. This study provides an efficient strategy to prepare cytotoxic protein, it provides a sample intuitive approach to probe the stability of protein dimerization too.