Abstract: Construction a nonspore B.subtilis 168 strain( B.subtilis 168Δspo0A) by deleting the spo0 A gene using the method of homologous recombination.The nonspore B.subtilis168Δspo0A strain was successfully got through PCR,nucleic acid electrophoresis,sporulation ratio and microscopy identification.The genome of this engineering strain does not need to insert resistance gene or any other selection marker gene after spo0 A genes' knockout which called clean deletion. This study is of great significance for industrial production of B.subtilis as well as improve safety of food and drug.