A genetically modified (GM) Bacillus thuringiensis strain WG-001 was constructed and prepared as a biopesticide for field trial of its biocontrol efficacy and for assessing the ecological impact on the indigenous microflora. Using a TnpI-mediated site-specific recombinant strategy, a cry1Ac10 and an additional p20 gene were transferred into B. thuringiensis wild-type strain YBT-1520 while all of the heterologous antibiotic resistance genes were selectively eliminated. The resultant engineered strain, WG-001, exhibited a two-fold increase of the total Cry proteins and an approximately 25% increase of lepidopterancidal potency in fermentation compared to its parent strain, and it was also given a distinct trait to allow tracking of its appearance using simple polymerase chain reaction verification of p20 and cry1-like genes. In the cotton field, released WG-001 biopesticide showed a limited aerial dissemination capability and dispersal range, and did not establish a population in the soils or on the cotton leaves of the field. There was no evidence supporting the significant biological influence of WG-001 on the indigenous microflora. These results demonstrate that the likelihood of risks of released WG-001 to non-target organisms in the cotton fields was limited by the low survival of the introduced strain.