The aerosolization characteristics of the fungal spores are affected by many environmental variables. The uniformity and distribution of spore concentrations on a fungal substrate is an important parameter for the stable and constant generation of fungal bioaerosols over long time periods. However, this traditional method using a light microscope is very slow and inefficient in evaluating the uniformity of spores on fungal substrates. In this study, we propose an evaluation method for spore concentration uniformity on fungal substrates using the real-time aerosolization characteristics of the fungal spores. A fungal bioaerosol generator, developed previously, was used for the steady aerosolization of fungal spores. The multiorifice air jets and the rotating substrate inside the generator were used to scan a fungal culture Petri plate and to aerosolize the fungal spores. Experimental tests on Cladosporium cladosporioides were conducted. The particle size distribution and total spore concentration of aerosolized fungal spores were measured using a particle-size distribution analyzer and a condensation particle counter. The results demonstrate that it is possible to evaluate the uniformity of the spore concentration of fungi on a substrate by measuring the variance of the particle concentration in real time. In addition, the uniformity of the fungal spore concentration has little effect on the particle size distribution under the selected experimental conditions.