The sensor-based monitoring of diseases under controlled conditions establishes an objective tool that allows a better understanding of the pathogen-plant interactions in different situations. The purpose of our work was to implement the presymptomatic detection of powdery mildew on wheat leaves shortly after fungus inoculation by spectral and time-resolved laser-induced fluorescence spectroscopy. In the general scope of plant phenotyping, we hypothesized that it is possible to discriminate between wheat genotypes that are either resistant or susceptible to powdery mildew. According to our results, the presymptomatic detection of powdery mildew on wheat leaves was accomplished, irrespective of genotype, as early as one day after inoculation using the fluorescence amplitude ratio F451:F522. Similarly, the ratios F451:F522, F522:F687, and F522:F736 of the half-bandwidth are also appropriate parameters. Furthermore, in the spectral range between 410 nm and 620 nm, the mean lifetime was significantly longer in inoculated leaves than it was in control leaves. Finally, the short-term (10-12 hour) increase of the fluorescence mean lifetime at 530 nm and 560 nm following the inoculation suggests that the speed of the plant reaction might be associated to its resistance to the pathogen. Based on this information, we conclude that determinations of ultraviolet, laser-induced fluorescence intensity and lifetime are suitable approaches to presymptomatically detect powdery mildew on wheat leaves one day after inoculation.