To improve the β-carotene content of rice endosperm, the synthesized phytoene synthase gene(psy) from maize and phytoene dehydrogenase gene(crtI) from Erwinia uredovora are transformed into rice under the control of rice endosperm-specific glutellin promoter. A single-copy inserted and marker-free psy/crtI homozygous transgenic line ky1-4 was selected by combining PCR screening with Southern blotting analysis. The flanking genomic sequence next to the right border of the T-DNA insertion site was determined by thermal asymmetric interlaced PCR (Tail-PCR). In the assistance of Southern blotting analysis, the insertion of ky1-4 was located on rice chromosome 11. The quantitative analysis by ultra-high performance liquid chromatography (UPLC) showed that β-carotene contents in seeds of different transformed lines ranged from (1.99±0.11) μg/g to (4.41±0.30) μg/g, and that of ky1-4 was (3.11±0.08) μg/g.