Due to the increasingly serious environmental pollution, it is necessary to trace the pollutants. The tracer Rhodamine B is an effective way to trace the source of pollutants. However, in the in-situ pollutant tracking and detection using Rhodamine B fluorescence sensor, the measurement results will be affected by environmental factors such as temperature and turbidity. Therefore, it is important that the accuracy of the in-situ detection of Rhodamine B is improved by compensating and correcting the two main environmental factors, temperature and turbidity. Fluorescence spectra of Rhodamine B with different concentrations were detected by fluorescence spectrophotometer, and partial least squares (PLS) method was used to analyze the spectral data and establish the standard curve. The fluorescence spectra of Rhodamine B were measured and analyzed in the range of temperature from 10 ℃ to 60 ℃ and turbidity from 0 NTU to 55 NTU. The results showed that the fluorescence intensity of Rhodamine B was negatively correlated with temperature and positively correlated with turbidity. Since the rate of change of Rhodamine B concentration difference has a good linear relationship with temperature and turbidity, the rate of change of Rhodamine B concentration difference is used for compensation correction in different environments. After temperature and turbidity compensation correction, the relative errors of concentration detection results were less than 0.48% and 0.34%, respectively, which improved the detection accuracy of Rhodamine B in different environments. What’s more, under the influence of temperature and turbidity, the detection results were analyzed, by doing so, a model of common compensation correction was established. It provides a correction method to suppress the interference of temperature and turbidity in-situ detection of Rhodamine B.