Incidence of iron (Fe) deficiency in human populations is an emerging global challenge. This study was conducted to evaluate the potential of iron sulphate combined with biochar and poultry manure for Fe biofortification of wheat grains in pH affected calcareous soil. In first two incubation studies, rates of sulfur (S) and Fe combined with various organic amendments for lowering pH and Fe availability in calcareous soil were optimized. In pot experiment, best rate of Fe along with biochar (BC) and poultry manure (PM) was evaluated for Fe biofortification of wheat in normal and S treated low pH calcareous soil. Fe applied with BC provided fair increase in root-shoot biomass and photosynthesis up to 79, 53 and 67%, respectively in S treated low pH soil than control. Grain Fe and ferritin concentration was increased up to 1.4 and 1.2 fold, respectively while phytate and polyphenol was decreased 35 and 44%, respectively than control in treatment where Fe was applied with BC and S. In conclusion, combined use of Fe and BC could be an effective approach to improve growth and grain Fe biofortification of wheat in pH affected calcareous soil.