Membrane-based filtration technologies have seen rapid inclusion in a variety of industrial processes, especially production of drinking water by desalination. Biological fouling of membranes is a challenge that leads to increased costs from efficiency reductions, membrane damage, and ultimately, membrane replacement over time. Such costs can be mitigated by monitoring and optimizing cleaning processes for better prognosis. A fluorescence-based sensor for early biofouling detection capable of measuring extracellular enzyme activity was developed. The selected fluorogen and fluorogen-substrate were characterized and down selected by in vitro screening for compatibility in seawater and profiled over relevant Red Sea desalination parameters (pH and temperature). ATP measurements are currently regarded as start-of-the-art when assessing biomass accumulation in membrane-based filtration systems Therefore, the fluorescence sensor response was measured for a range of bacterial concentrations and validated using an ATP assay. We demonstrate the efficacy of the proposed approach for the quantitative assessment of bacteria activity in seawater rapidly and sensitively. Following in vitro testing, the method was employed in a lab-scale seawater reverse osmosis (SWRO) system for suitability in monitoring biofouling formation. The sensor successfully measured bacterial biomass accumulation rapidly and non-invasively using exogenously applied fluorogen-substrates. The sensor response was corroborated with real-time in situ non-destructive imaging of the membrane surface. This approach demonstrates the practicality of prototyping an early-detection biofouling sensor in membrane based processes using extracellular enzyme activity as a measure of bacterial abundance.