Abstract <p><i>Bacillus</i> strains are widely used in food fermentation and plant disease control. An efficient transformation method is crucial for genetic manipulation in these organisms. To enhance the transformation efficiency of three <i>Bacillus</i> strains—<i>Bacillus amyloliquefaciens</i> YN-J3 (B.a YN-J3), <i>Bacillus velezensis</i> JN-Y2 (B.v JN-Y2) and&#xa0;<i>Bacillus subtilis</i> S-16 (B.s S-16), we optimized transformation conditions using orthogonal experiments combined with response surface analysis. Additionally, we tested various cell wall agents to improve competence. Our results showed that the optimal transformation parameters for B.a YN-J3 and B.v JN-Y2 had an OD₆₀₀ of 0.70, a competent cell volume of 91 μL, a plasmid concentration of 1040&#xa0;ng·μL⁻¹, and a field strength of 18.1&#xa0;kV·cm⁻¹. For B.s S-16, the optimal conditions were an OD₆₀₀ of 0.71, a competent cell volume of 92 μL, a plasmid concentration of 1052&#xa0;ng·μL⁻¹, and a field strength of 18.2&#xa0;kV·cm⁻¹. Under these optimal conditions, the transformation efficiencies for B.a YN-J3, B.v JN-Y2, and B.s S-16 were 22,198.33&#xa0;CFU·μg⁻¹ DNA, 24,498.67&#xa0;CFU·μg⁻¹ DNA, and 23,305.00&#xa0;CFU·μg⁻¹ DNA, respectively. Screening of cell wall agents revealed that 50&#xa0;mg/mL glycine significantly boosted transformation efficiency by 40, 36, and 24 times for B.a YN-J3, B.v JN-Y2, and B.s S-16, respectively. These findings demonstrate that combining glycine treatment with optimized transformation conditions provides an efficient approach for the genetic manipulation of <i>Bacillus</i> strains.</p> Key points <p>• <i>The electroporation transformation parameters of three Bacillus were optimized by combining orthogonal experiments with response surface methodology</i></p> <p>• <i>A stable and efficient electroporation transformation system suitable for three types of Bacillus was established.</i></p> <p>• <i>3. 50 mg/mL glycine solution can increase the transformation efficiency by 40, 36 and 24 times, respectively.</i></p>