The most widely used method of identification of microbial morphology and structure is microscopy, but it can be difficult to distinguish between pathogens with a similar appearance. Existing fluorescent staining methods require a combination of a variety of fluorescent materials to meet this demand. In this study, unique concentration-dependent fluorescent carbon dots (CDs) were synthesized for the identification and quantification of pathogens. The emission wavelength of the CDs could be tuned spanning the full visible region by virtue of aggregation-induced narrowing of bandgaps. This tunable emission wavelength of the specific concentration response to diverse microbes can be used to distinguish microorganisms with a similar appearance, even in a same genus. A hyperspectral microscopy system was demonstrated to distinguish Aspergillus flavus and A. fumigatus based on the results above. The identification accuracy of the two similar-looking pathogens can be close to 100%, and the relative proportions and spatial distributions can also be profiled from the mixture of the pathogens. This technique can provide a solution to the fast detection of microorganisms and is potentially applicable to a wide range of problems in areas such as healthcare, food preparation, biotechnology, and health emergency.