Both excitation and single emission of Bi24Al2O39:Mn5+ phosphors are possible in the near-infrared (NIR) region, which can be simultaneously detected. Monitoring the excitation radiation allows us to prevent its fluctuations from affecting the luminescence. Single emission is a technique that involves the luminescence intensity ratio, so fluctuations in the intensity of the excitation radiation do not affect the result. This technique has the advantage of involving conventional methods and is not affected by optical dispersion, so it provides reliable results suitable for practical applications. Using this method, we investigate the temperature-sensing characteristics of Bi24Al2O39:0.5%Mn5+ phosphors from 300 to 380 K. Under 970 nm excitation, the maximal relative sensitivity is 3.54% K−1 in the physiological temperature range (300–330 K) and reaches 3.98% K−1 at 351 K. The excellent temperature sensing and NIR excitation and emission suggest that this NIR thermometer can be used for biological applications.