An efficient and reproducible Agrobacterium tumefaciens-mediated transformation method was developed for Gossypium hirsutum L. 'KC3' using a shoot apex explant with the combination of microinjection and sonication. To optimize the transformation efficiency, preculture conditions, acetosyringone concentration, A. tumefaciens cell density, and precision microinjection with sonication were evaluated. A high-throughput A. tumefaciens-mediated genetic transformation method was developed using the uidA gene. The shoot apex explants were microinjected with an A. tumefaciens strain EHA105 carrying the uidA gene and co-cultivated for 3 d on mMS medium fortified with 100 mM acetosyringone. Among the different parameters and conditions tested, preculturing explants for 24 h, microinjecting three times with an A. tumefaciens cell suspension of OD600 nm = 0.6 containing 100 mM acetosyringone, and sonicating explants for 20 s produced the maximum transformation efficiency of 20.25%. Putative transformants were transferred to regeneration medium supplemented with 0.8 mg L-1 1-naphthaleneacetic acid, 2 mg L-1 thidiazuron, 16% (v/v) of an Oscillatoria acuminata extracellular extract, and 50 mg L-1 kanamycin. Initially, the transgenic shoots were evaluated by GUS histochemical analysis, and integration was confirmed by polymerase chain reaction and Southern blot analysis. Plantlets transferred from an artificial growth chamber to greenhouse conditions showed a higher survival rate of 90% compared to the directly transferred plantlets. This simple and efficient transformation system could be valuable to transfer any gene of interest into cotton with high transformation efficiency in a short time.