In situ rapid characterization of river autotrophic biofilms may be very useful for the monitoring of small to medium rivers in which periphyton represents most of algal biomass. In a water column, fluorimetric probes have demonstrated their ability to immediately estimate the chlorophyll a concentration and the relative proportion of different autotrophic groups in the phytoplankton biomass (Diatoms, Green Algae, Cyanobacteria and Cryptophytes). In this study, the possibility to use a fluorimetric probe (Fluoroprobe bbe) on periphyton was evaluated either after biofilm resuspension in an algal-free water volume or via the use of an adapter (Benthofluor bbe) which allows a direct measurement on a substrate. The probe was tested with natural biofilm maintained in the laboratory and with artificially generated biofilms from monospecific cultures. Chl a estimations by the probe were generally well correlated to standard spectrophotometric determinations. However with the Benthofluor, a saturation of the signal was observed for chl a densities in the biofilm above 0.7 µg.cm-2 (thickness effect). Furthermore chl a content was underestimated after biofilm resuspension (shadow effect of aggregates) and rather overestimated with the Benthofluor (matrix effect). The probe showed high repeatability of the measurements and high sensitivity with the Benthofluor especially. Making non destructive measurements, the latter appears well suited for establishing periphyton growth kinetics on artificial substrates, either in situ or in laboratory. Experimentally, a faster periphyton growth was observed at higher nutrient concentrations with the Benthofluor. Some field measurements made on stones confirmed that the device is a promising tool for monitoring eutrophication. If the probe could quite well recognize the algal groups present in a biofilm after its resuspension, the determination of the relative biomasses was altered with this way of proceeding. Directly applied onto the substrate, the probe completely failed to discriminate the algal groups. It seems that the calibration of the Benthofluor has to be improved in order to obtain reliable phytobenthic compositions of biofilms.