Abstract: BOD₅ detection is a microbial-based pollution load assessment, there are many articles demonstrating that correlation between the fluorescence intensity of microbial metabolite (tryptophan-like) and BOD₅ measurement. The relationship between the fluorescence intensity and BOD₅ value of surface water samples in Changzhou section of the Yangtze River Basin was compared and verified. Turbidity, pH, electrical conductivity and regional differences were investigated to evaluate the potential of fluorescence method for BOD₅ pollution assessment. It was found that the pH and ion interference of surface water had no significant effect on the detection of BOD₅ by fluorescence method. The maximum value of t was 1.227, which no significant effect. Compared with the standard method, the correlation coefficient of fluorescence method r was 0.909 9, which had obvious correlation. The results showed that the fluorescence method has few influencing factors, fast detection speed, high degree of automation, and stable and reliable results when applied to BOD₅ detection in surface water, and can be used as an alternative method for BOD₅ detection and the main means to characterize the pollution degree of biodegradable pollutants in water.