Modern tissue culture and genetic transformation techniques play a significant role in manipulating biosynthetic pathways and enhancing the production of secondary metabolites in medicinal plants. Although a standardized method for the genetic transformation of Hyoscyamus niger has not yet been established, by optimizing factors influencing Agrobacterium tumefaciens-mediated transformation—one of the most effective methods for genetic modification—it is possible to increase the frequency of regeneration and transformation in plant cells. In this study, with the aim of developing an efficient protocol to improve gene transfer efficiency in H. niger, three main factors were evaluated: the A. tumefaciens strain (EHA105, GV3101, and LBA4404), explant type (leaf and petiole), and transformation method (agroinoculation, agroinfection, agroinjection, and agrosonication). Optimization of gene transfer conditions revealed that petiole explants inoculated with the LBA4404 strain using agrosonication and agroinoculation methods exhibited the highest rates of callus and embryo formation, respectively. However, histochemical gusA staining indicated that among the hygromycin-resistant plantlets, only those derived from leaf explants inoculated with the LBA4404 strain carrying the pCAMBIA1304 vector via agroinfection showed positive X-gluc staining and contained blue pigments, achieving the highest transformation rate at 21.74%. The successful integration of gusA and hptII genes in putative transgenic plantlets was confirmed through PCR and RT-PCR analyses. Therefore, by investigating key factors affecting A. tumefaciens-mediated transformation, an efficient genetic transformation protocol for H. niger can be established.