Microbial aerosol surveillance is essential to ensure national security and human health. The online monitoring method based on fluorescence detection technology stands out in microbial aerosol monitoring due to its advantages of fast response, strong portability, and high sensitivity. This method accurately identifies individual bioaerosol particles and monitors real-time aerosol content in the environment. Its working principle is to use ultraviolet lasers to induce the intrinsic fluorescence of bioparticles and then detect the intrinsic fluorescence signals to achieve the identification of bioparticles. Although fluorescence online monitoring equipment has significant advantages in monitoring the concentration and particle size of biological particles in ambient air, the technological development in this field in China is still in its infancy. In the past 20 years, significant progress has been made in laser-induced online monitoring of microbial aerosols. These advances are mainly reflected in the optimization of excitation light cost, the subdivision of detection sorting channels, and the advancement of robotics and algorithm programming languages. However, the quantitative application of these instruments is limited by the lack of standard fluorescence calibration methods, which also increases the complexity of comparing different measurements. With the widespread application of online monitoring instruments, fluorescence aerosol detection instruments' performance evaluation and calibration technology have gradually attracted attention. The calibration research on fluorescence bioaerosol detection systems is still in its infancy, and no unified performance evaluation criteria have yet been formed. This paper briefly introduces the development of fluorescence online monitoring instruments, the research status of fluorescence-based single-particle online monitoring, and its calibration technology, which is summarized for the first time. It provides strong support for developing microbial aerosol detection technology and establishing a performance evaluation system for microbial aerosol detection instruments.