Alnus glutinosa is an important woody plant in Lithuanian forest ecosystems. Knowledge of fluorescence properties of black alder pollen is necessary for scientific and practical purposes. By the results of the study, we aimed to evaluate possibilities of identifying Alnus glutinosa pollen fluorescence properties by modeling ozone effect and applying two different fluorescence-based devices. To implement the experiments, black alder pollen was collected in a typical habitat during the annual flowering period in 2018–2019. There were three groups of experimental variants, which differed in the duration of exposure to ozone, conditions of pollen storage before the start of the experiment, and the exposure time. Data for pollen fluorescence analysis were collected using two methods. The microscopy method was used in order to evaluate the possibility of employing image analysis systems for investigation of pollen fluorescence. The second data collection method is related to an automatic device identifying pollen in real time, which uses the fluorescence method in the pollen recognition process. Data were assessed employing image analysis and principal component analysis (PCA) methods. Digital images of ozone-exposed pollen observed under the fluorescence microscope showed the change of the dominant green colour toward the blue spectrum. Meanwhile, the automatic detector detects more pollen whose fluorescence is at the blue light spectrum. It must be noted that assessing pollen fluorescence several months after exposure to ozone, no effect of ozone on fluorescence remains.