For about 10 years, environmental tracing development using dissolved organic matter (DOM) has been the subject of several studies. Particularly, the use of characterization techniques, like fluorescence emission-excitation matrices has enabled the identification of DOM sources and monitoring them within mainland or marine hydrosystems. Moreover, hydrogeologists have shown the significance of total organic carbon content used as a fast seepage tracer in karstic aquifers. The aim of this study consists in using DOM fluorescence signals to develop a transit time semi-quantitative tracer in heterogeneous hydrosystems. The Low-Noise Underground Laboratory (Vaucluse, France) cuts the network of Fontaine de Vaucluse (FV) karstic vadose zone randomly, and offers a special access to different unstructured dripwaters, with different hydrodynamic behaviour, inside its galleries, i.e. not hierarchical as in natural caves. Previous long-term hydrodynamic and hydrochemical studies allowed the understanding of their hydrogeological behaviour and the estimation of mean transit times. That is why this site is adequate to develop new transit time tracers. After identification of the different DOM sources (i.e. lithic and rendzic leptosols), fluorescence intensities monitoring from soil leachates and dripwaters, for certain excitation-emission wavelength pairs, allowed the development of punctual transit time tracing, by spotting infiltration periods of fluorescent compounds, and monitoring their transfer within a hydrosystem. A fluorescence index (humification index) and the mean transit time of each gallery groundwater, stemmed from previous hydrodynamic and hydrochemical studies, allowed the calibration of a logarithmic relationship. This one allows the development of a continuous transit time tracing method that estimates transit times without long-term studies. It has been tested on two springs of FV catchment basin, providing transit time estimations for karstic hydrosystems that do not present a mixture between recent and pluriannual waters.