Iron (Fe) and zinc (Zn) deficiencies severely threaten global human health. Thus, rice biofortification to enhance intrinsic Fe and Zn levels in grains represents an effective strategy to alleviate human Fe and Zn deficiencies. Several biofortification strategies have successfully increased Fe and Zn concentrations in the rice endosperm, with multigene approaches demonstrating synergistic micronutrient accumulation. To enhance Fe and Zn accumulation in rice endosperm, we designed a transformation construct (NYFN) in which OsNRAMP7 and OsNAS2 were driven by the 35S promoter, OsYSL2 by the OsSUT1 promoter, and OsFER2 by the endosperm-specific OsGluA2 promoter. Multi-year field trials were conducted at two locations to evaluate Fe and Zn accumulation in transgenic NYFN rice plants derived from Nipponbare (NB) and commercial cultivar Huaidao 5 (HD5) genetic backgrounds. The results showed that transgenic NB lines exhibited 10.93-14.72 μg/g DW Fe and 33.01-48.33 μg/g DW Zn in polished grains, representing 4.9- to 6.3-fold increases in Fe and 2- to 2.7-fold increases in Zn compared to the NB control. Polished grains of HD5 transformants contained 10.24-13.35 μg/g DW Fe and 32.17-50.33 μg/g DW Zn, corresponding to 4- to 7.4-fold elevations in Fe and 2- to 2.3-fold elevations in Zn relative to the HD5 control. X-ray fluorescence spectroscopy (µ-XRF) and Perls' Prussian blue staining analyses confirmed the significantly enhanced Fe and Zn accumulation in transgenic grains. Transgenic NB lines exhibited significant changes in certain agronomic traits, including reduced 1000-grain weight, grain size, and grain filling rate, whereas transgenic HD5 lines showed no significant agronomic differences relative to the wild type. Total grain weight per plant remained unchanged in both the transgenic NB and HD5 lines compared to the wild type. The results demonstrate that the NYFN strategy enables sustainable Fe and Zn biofortification, representing a promising solution to the global challenge of human Fe and Zn deficiency.