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Further improving the quality of surface water is becoming more difficult after the control of main point-sources, especially in the complex pollution area with mixed industrial and agricultural productions, whereas the pollution source apportionment might be the key to quantify different pollution sources and developing some effective measures. In this study, a technical framework for source apportionment based on three-dimensional fluorescence and microbial traceability model is developed. Based on screening of the main environmental factors and their spatiotemporal characteristics, potential pollution sources have been tentatively identified. Then, the pollution sources are further tested based on the analysis of fluorescence excitation-emission matrix (EEM) and the similarity of fluorescence components in surface water and potential pollution sources. At the same time, the correlation between microbial species and pollution sources is constructed by analyzing the spatiotemporal characteristics of microbial composition and the response of main species to environmental factors. Therefore, pollution source apportionment is quantified using PCA-APCS-MLR, Fast Expectation-maximization for Microbial Source Tracking (FEAST), and Bayesian community-wide culture-independent microbial source tracking (SourceTracker). PCA-APCS-MLR could not effectively distinguish the contributions of different industrial sources in the complex environment of this study, and the contribution of unknown sources was high (average 39.60%). In contrast, the microbial traceability model can accurately identify the contribution of 7 pollution sources and natural sources, effectively reduce the proportion of unknown sources (average of FEAST is 19.81%, SourceTracker is 16.72%), and show better pollution identification and distribution capabilities. FEAST exhibits a more sensitive potential in source apportionment and shorter calculation time than SourceTracker, thus might be used to guide the precise regional pollution control, especially in the complex pollution environments. [Display omitted] • Main pollution sources in distinct areas are proposed via the source apportionment. • Constructing the correlations of fluorescence components and microbes via biomarkers. • Fluorescent fingerprints coupled with microbes enhance pollution apportionment accuracy. • FEAST is more accurate than SourceTracker for complex pollution sources apportionment. [ABSTRACT FROM AUTHOR]