Abstract: The key to increasing the accuracy of isotope measurement is the proper mass fractionation of instrumental mass discrimination calibrated by double spike. This paper took Mo isotopes as an example to introduce the principle, illustrate the calculation method of high-precision measurement isotope composition with double spike, and demonstrate the analytical method with multi-collector inductively coupled plasma-mass spectrometer(MC-ICP-MS). The ¹⁰⁰Mo/⁹⁷Mo of double spike and standard were calibrated by ¹⁰⁴Pd/¹⁰²Pd of Pd standard solution, and other ratios of double spike and standard were calibrated by ¹⁰⁰Mo/⁹⁷Mo. On the premise of that the instrument fractionation and the nature fractionation follow an exponential law, a system of nonlinear equations was converted to a system of linear equations using the Taylor formula, and the fractionation factor of the sample was calculated by the Newton iterative method. δ¹⁰⁰Mo/⁹⁵Mo, δ⁹⁸Mo/⁹⁵Mo and δ⁹⁷Mo/⁹⁵Mo were measured 20 times and averaged when analyzing with MC-ICP-MS.