In this manuscript we describe a sensitive, fixed-wavelength, lock-in-based fluorescence detector built from a light-emitting diode (LED), two colour filters, a photodetector, a small number of discrete analogue components, and a low-cost microcontroller development board. We describe the construction, operating principle, use and performance of the fluorescence detector, which allows measurements to be carried out in a 10-mm pathlength cuvette or a low volume (< 100 l) flow-cell. For illustrative purposes the detector is applied here to a cholesterol assay based on the enzyme-mediated conversion of (nonemissive) Amplex Red into the fluorescent dye resorufin. The system may be readily adapted to other biomolecules through a simple change of enzyme.