The increasing use of modified Fe3O4 magnetic microparticles has raised safety concerns regarding their use and effect on human health. This study assessed the in vivo biosafety, DNA, and chromosome damage of modified Fe3O4 microparticles such as Au@Fe3O4, Ag@Fe3O4, Cs@Fe3O4, Pt@Fe3O4, and CdS@Fe3O4, using spleen-deficient rats. Spleen-deficient rats treated with naked and modified (Au, Cs, Pt) Fe3O4 microparticles (5000 mg/kg) displayed low toxicity. Only treatment with Cds@Fe3O4 resulted in elevated toxicity and death in rats. Au-, Ag-, and Pt-modified Fe3O4 increased the rate of hemolysis in rats relative to treatment with naked Fe3O4. Despite this, Au- and Pt-modified Fe3O4 increased the biocompatibility and reduced DNA and chromosome damage in rats relative to naked Fe3O4. While Cs@Fe3O4 microparticles displayed a higher biocompatibility than naked Fe3O4, they displayed no significant reduction in DNA and chromosome damage. In summary, Au and Pt surface-modified Fe3O4 microparticles display elevated in vivo biosafety compared to unmodified particles. The precious metal material, with good biological compatibility, surface modification of Fe3O4 is an effective strategy to improve the overall safety and potential therapeutic utility of these magnetic materials.