The iron isotope composition of iron oxides such as magnetite has been widely used to study petrogenesis and ore genesis. In situ Fe isotope compositions of iron oxides are commonly determined using laser ablation multi-collector inductively coupled plasma mass spectrometry (LA-MC-ICP-MS) due to its high spatial resolution and rapid analytical procedure. The pure iron IRMM-014 is commonly used as an international Fe isotopic reference standard. However, some studies have shown that there will be a significant matrix effect during the Fe isotope analysis of magnetite when using IRMM-014 as the calibration standard. Here, a high-temperature and high-pressure (500 °C and 1.2 GPa) sintering method was used to prepare magnetite (MtFe-1) with homogeneous Fe contents and Fe isotope compositions. The magnetite tablet was characterized by laser Raman spectroscopy and an electron probe microanalyzer to assess the homogeneity of crystal structure and major and minor elements. In situ Fe isotope analysis across the magnetite tablet determined by femtosecond LA-MC-ICP-MS showed good homogeneity, with a standard deviation (external precision) of 0.08–0.09‰ (2SD, n = 38) for the δ⁵⁶Fe value and 0.15–0.16‰ (2SD, n = 38) for the δ⁵⁷Fe value. Solution MC-ICP-MS analyses from three independent laboratories yielded average δ⁵⁶Fe_IRMM-014 and δ⁵⁷Fe_IRMM-014 values of 0.34 ± 0.03‰ (2SD, n = 8) and 0.50 ± 0.05‰ (2SD, n = 8) for MtFe-1, respectively. A natural magnetite Mgt 08BI-12 with a known Fe isotope composition was determined in situ using MtFe-1 as the bracketing standard, which yielded an Fe isotope composition that is consistent with the results of the solution method. In summary, MtFe-1 can be used as a bracketing standard for in situ Fe isotope analysis using LA-MC-ICP-MS and the application of the Fe isotope tracer in earth science research will be extended.