In this study, a simple and stable genetic transformation system of foxtail millet (Seteria italica) was established and optimized, in which shoot tips were used as the explant. We transformed Yugu 1, an elite millet cultivar, by Agrobacterium-mediated transformation, and tested different treatments to boost transformation efficiency. We used a PCR-based assay to screen transformants in third-leaf stage seedlings. We determined an optimal lethal concentration of glufosinate (Basta) when sprayed to millet seedlings, and tested the different PCR-based genotyping methods with or without Basta spary. Using the newly established pipeline, we further optimized various crucial factors that affect genetic transformation efficiency. We found that an optimal concentration of bacterial culture was OD₆₀₀=1.4, an optimal concentration of acetolsyringone was 800 μmol∙L ^-1. We also obtained high transformation efficiency with an infecting pressure at 0.05 MPa, and an infecting time of 40 min. We used the above-mentioned transformation method to transform a Seteria italica calcineurin B-like protein 4 (SiCBL4) overexpression construct. Genetic stability analysis on T₂ generation transformed plants was performed by the combination assay of Basta resistance and real-time quantitative fluorescence RT-PCR, which can save the time of genotyping. Altogether, this study establishes a shoot tip-based stable genetic transformation system for foxtail millets, and also develops a robust pipeline to detect transgenic offsprings.