Currently it is still extremely difficult to adequately sample populations of microalgae on sediments for large-scale biomass determination. We have now devised a prototype of a new benthic sensor (BenthoFluor) for the quantitative and qualitative assessment of microphytobenthos populations in situ. This sensor enables a high spatial and temporal resolution and a rapid evaluation of the community structure and distribution. These determinations are based on the concept that five spectral excitation ranges can be used to differentiate groups of microalgae, in situ, within a few seconds. In addition, because sediments contain a lot of yellow substances, which can affect the fluorescence and optical differentiation of the algae, the device was equipped with a UV-LED for yellow substances correction. The device was calibrated against HPLC with cultures and tested in the field. Our real-time approach can be used to monitor algal assemblage composition on sediments and is an ideal tool for investigations on the large-scale spatial and temporal variation of algal populations in sediments. Apart from the differentiation of algal populations, the BenthoFluor allows instantaneous monitoring of the chlorophyll concentrations and determination of which algae are responsible for this on the uppermost surface of sediments in the field and in experimental set-ups. © 2006 E. Schweizerbart'sche Verlagsbuchhandlung.