【Objective】 The hygromycin resistance gene is often used as a selection marker in expression vectors for multiplication of common genic male sterile (CGMS) lines in rice. The glyphosate resistance gene replaces the hygromycin resistance gene as it not only gives the multiplication line glyphosate resistance, but also bypasses the current policy restrictions on the use of antibiotic marker genes in genetically modified crops. 【Methods】 An expression vector pC3300-Epsps-AA-Eat-Red was constructed by using homologous recombination method in order to multiply the rice CGMS line with eat1 mutation gene, which harbors the glyphosate resistance gene Epsps^#, pollen lethal gene ZmAA1, CGMS restorer gene OsEat1 and red fluorescent gene DsRed2. Agrobacterium-mediated transformation method was adopted. 【Results】 Transformation of indica rice line 9K19-5 produced 318 regenerated plants of the CGMS multiplication line Eat9K that could restore the sterility of eat1 and resist glyphosate. The phenotype identification of single copy transformant Eat9K-3 showed that: there were two kinds of pollens in anthers, one was fertile and the other was sterile; the seeds included two types, one was with fluorescence and the other was without fluorescence; the seedlings from fluorescent seeds tolerated at least 1 g/L glyphosate. In addition, this study established a four-primer detection method to distinguish the wild-type gene Eat1 from the genic male sterile gene eat1. 【Conclusion】 This study not only verified the effectiveness of the expression vector, but also created the new germplasm of CGMS multiplication line with herbicide resistance, as well as established a four-primer detection method to distinguish the wild-type gene Eat1 from genic male sterile gene eat1.