Measurement of the sample temperature in biophysics research is challenging, as the samples are commonly placed in a miniature sample chamber under a microscope. In this study, we proposed a method to measure the temperature of an aqueous solution in miniature sample chambers in a microscopic system. Existing studies show that the absorption coefficient spectrum of water shifts with temperature, especially in the near-infrared (NIR) band. We measured the absorption spectra of water with different temperatures and analyzed them, to build a mathematical model relating the temperature and the spectrum. A setup for temperature measurement in a microscopic system was designed and implemented by coupling a spectrometer and a light source to a microscope. The temperature could be calculated by the spectral data and the mathematical model while simultaneously observing the micro-image of the sample. A series of liquid samples at different temperatures were tested using the setup, and the root mean square error of the calculated temperature is less than 0.5 °C. The results demonstrate that the method based on the NIR spectrum can be used for noncontact and quick measurement of the liquid sample temperature in a microscopic system.