Magnetic nanoparticles (MNPs) have excellent magnetic-temperature characteristic. However, current temperature measurement based on MNPs is interfered by concentration. Utilizing the electron spin resonance (ESR), we propose a highly sensitive temperature measurement method without concentration coupling. The anisotropic field is affected by temperature, thus affecting the g-value. The influence of the MNP concentration, size, and the data analysis method on temperature estimation are studied. The optimal temperature sensitivity is achieved with 15-nm MNPs while Gaussian smoothing method allows an optimal accuracy at Fe concentration of 5 mg/ml with a root mean squared error of 0.07 K.