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. Monitoring bacterial accumulation in situ can therefore advance understanding of cleaning efficiency. A fluorescence-based sensor for early biofouling detection capable of measuring extracellular enzyme activity was developed and tested in a lab-scale seawater reverse osmosis (SWRO) biofouling model for use in monitoring bacterial accumulation proximal to the surface of a membrane. We tracked bacterial biomass accumulation rapidly and non-invasively using exogenously applied fluorogen-substrates and corroborated with optical coherence tomography imaging of the membrane surface in real-time. The selected fluorogen and fluorogen-substrate were characterized and down selected by high throughput screening in vitro for compatibility in seawater and profiled over relevant Red Sea desalination parameters (pH and temperature). This approach demonstrates the practicality of prototyping an early-detection biofouling sensor in membrane based processes, such as seawater desalination, using extracellular enzyme activity as a measure of bacterial abundance.