The invention discloses a method for in-vivo temperature measurement based on AC magnetization of magnetic nanoparticles, and relates to a nano test technology field. The invention positions magnetic nano agent at an area of a measured object, applies an AC excitation magnetic field to the area of the magnetic nano agent, collects an AC magnetization signal of the magnetic nano agent under the AC excitation magnetic field, detects amplitudes of odd harmonics of the AC magnetization signal, and finally calculates in-vivo temperature according to a relationship between the odd harmonics and the in-vivo temperature. The invention predetermines the relationship between the odd harmonics and the in-vivo temperature via the discrete Langevin's function and the Fourier transformation, solves the in-vivo temperature according to the relationship without considering concentration of the magnetic nanoparticles, and effective moment as temperature varies, and thus accurately detecting the in-vivo temperature.