Developing flocculants with sterilization and monitoring functions is of practical significance for optimizing water treatment agents. In this research, a fluorescent monomer, EtFl, with double bonds was synthesized and copolymerized with acrylamide (AM) and 3-(methacryloyloxy)propyltrimethylammonium chloride (MAPTAC) using a linear anionic template agent. This synthesis successfully produced a multifunctional template copolymer, TP(AM-MAP-EtFl), with flocculation, sterilization and monitoring capabilities. The fluorescence intensity of TP(AM-MAP-EtFl) was measured using a fluorescence spectrophotometer, demonstrating strong performance even with an EtFl monomer mass fraction as low as 0.5 wt‰, resulting in a fluorescence intensity value of 1000 a.u., suitable for analytical detection. In flocculation experiments with simulated wastewater containing E. coli, the introduction of the fluorescent monomer did not affect E. coli flocculation. When flocculation conditions are optimized, the OD600 readings of the clear liquid remaining after treating with TP(AM-MAP-EtFl#0.5) and TP(AM-MAP-EtFl#5) are 0.0297 and 0.0457, respectively, illustrating superior sterilization effects. Interestingly, SEM and 3D-EEM results confirmed TP(AM-MAP-EtFl)’s bactericidal effect on E. coli, with the introduction of the fluorescent monomer not affecting the sterilization capability of quaternary ammonium. This work integrates flocculation, sterilization and fluorescence properties to provide sterilization-type flocculants with monitoring capabilities, enabling accurate calculation of polymer concentration by detecting solution fluorescence intensity, thereby offering potential for online detection and automatic control of water treatment systems, demonstrating good applicability.