Assimilable organic carbon (AOC) is recognized as an important parameter to evaluate the biostability of water. Studies have been carried out to investigate the easier and faster AOC detection methods in recent years. In our study, the relationship between AOC and excitation-emission matrix (EEM) was investigated through analysis of wastewater from a coal chemical industrial corporation, including biochemical effluent, ultrafiltration effluent, and reverse osmosis concentrate. Considering the influence of water sample properties on AOC distribution, these water samples were fractionated according to their hydrophilicity and acid/base properties. Neutrals and hydrophobic acids were major components of total organic carbon and AOC concentration of these fractions was measured. EEM spectra of water samples were divided into five regions according to fluorescence peaks. Distribution of fluorescence region integration (FRI) of water samples was also calculated, as well as other fluorescence parameters. Statistical analysis showed that the concentration of AOC presented high positive correlation with the FRI in region H2, with R2 = 0.696. Monte Carlo simulation also proved that the proportion of significant R2 (p < 0.05) was high at 89.1%, suggesting that the model was reliable at least at the qualitative level. In that case, FRI in Region H2 could be an indication for AOC concentration in water samples. Our findings focus on fundamental insights into establishing relationship between spectroscopy method and AOC in wastewater and provide an easier way of accessing AOC in coal chemical industrial wastewater. Further investigation could be oriented to the dynamic analysis of AOC transformation and tracing.