Abstract: The temperature of extracellular fluid is a significant characteristic for electrophysiological experiments of patch clamp. It is important to understood the factors affecting the temperature measurement of an extracellular fluid, such as the effect of the concentration of different ions. In this study, the effect of different ions at different concentrations in a small amount of physiological solution on the measurement accuracy of a temperature model was investigated by near-infrared spectroscopy and chemometrics. The interval partial least squares method was used to select an effective wavelength range and develop calibration models using the spectra in the selected range and temperature values. The results indicate that Ca2 has the greatest effect on the measurement accuracy of the temperature model of a cell physiological solution, followed by K , and Na has the least effect. Moreover, the results indicate that the model accuracy decreased with the increase in ion concentration. These findings provide a basis for correcting the effects of different ion concentrations in a physiological solution for improving the accuracy of temperature measurements by near-infrared spectroscopy.