<p><i>Coix lacryma-jobi</i> L. is a widely cultivated crop in East and Southeast Asia that has high nutritional value and medicinal activity. However, the traditional breeding process is long and tedious, with low breeding efficiency. To improve the efficiency of germplasm innovation, a stable and efficient shoot tip genetic transformation system was established in vitro using glyphosate Basta as the selective agent and Coix seeds of the “Xingrenxiaobaike” variety as the experimental material. By means of the <i>Agrobacterium tumefaciens</i>-mediated shoot tip genetic transformation method, combined with Basta and PCR to identify transgenic plants, several important factors affecting the shoot tip genetic transformation efficiency were discussed, and the conditions of the transformation system were explored and optimized. The results showed that the optimal concentration for Basta spraying treatment in screening transgenic Coix seedlings was 100&#xa0;mg/L. The concentration of <i>Agrobacterium</i> resuspended bacterial solution (OD₆₀₀) was 0.80, the infection pressure was 0.055&#xa0;MPa, the infected and only exposed stem tip meristem tissue was infected for 15 min, and the use of seedling sponge during dark cultivation was beneficial for the genetic transformation of the infected plant, with an average transformation rate of about 6.67%. Simultaneously, the above optimization system was used to obtain the p9286 ubi <i>Bar:: GUS</i> gene transgenic plants of Coix, and the transgenic situation was detected by spraying Basta and PCR. The droplet digital PCR results of the T0-generation transgenic plants and the GUS staining activity of the T1 generation seedlings were verified, proving that the transgenic plants can successfully pass on foreign genes to their offspring.</p>