The ability to analyze biotoxicity of atmospheric pollution plays an important role in public health. It provides the potential to directly analyze the health information of at-risk individuals. Although air quality standards have received significant attention in many countries, the potential for better biotoxicity assessment has remained largely unexplored. Here we propose a method using one kind of luminescent bacterium Photobacterium phosphereum to detect the biotoxicity of atmospheric particulate matter <= 2.5 mu m (PM2.5). Combined with the results of air pollution data of the year 2013-2014, this method has been proven to have good biotoxicity detection performance, and can evaluate the severity of at least 85% of PM2.5 related biotoxicity in Shanghai during this time period. Based on an established algorithm of this detection system, the biotoxicity of twelve PM2.5 real samples (collected over a month) were tested and divided into different biotoxicity levels. It allows an effective evaluation of biotoxicity of PM2.5 due to the quick and sensitive response of bioluminescence to the concentration of toxic components, which provides a valuable reference to evaluate the biotoxicity of PM2.5. This established method can be easily applied to the analysis and evaluation of any other PM2.5 samples assay by following the steps.