Air pollution has become one of major environmental challenges facing mankind in this century. However, current atmospheric toxicity studies are most offline with low sensitivity and limited understanding of collective biological responses to ambient stimuli on the single cell level. Here, we developed an online analysis system named as SLEPTor (Single Living yEast PM Toxicity Sensor) which allows us to analyze aerosol particle toxicity in vivo. The SLEPTor is composed of an automated air sampling, microfluidics, GFP(green fluorescent protein)-tagged Saccharomyces cerevisiae yeast sensor as well as multiplexing automated fluorescence imaging system. In testing the SLEPTor, 63 yeast genes were screened simultaneously here for their responses to both manmade reactive oxygen species (ROS) and also Beijing aerosol samples. For ROS samples, MRS3, MRS4, BSD2 and ZRC1 proteins were detected in increased abundances, and the BSD2 protein expression, related to oxidative stress, was more pronounced, followed by MRS4 (for DNA repair) with a peak expression after 20 min exposure. In contrast, HSP60 (oxidative stress), SSA1 (DNA repair) and MSB1 (MAPK pathway) proteins were observed to have elevated expressions visualized with time-lapsed movies when challenged with Beijing air samples (150 pg/ms) for 100 min. The same experiments were repeated twice involving 63 genes using the SLEPTor and further confirmed by a spectra method. Among the screened proteins, SSA1 was found to be sensitive to ambient air. Using the SLEPTor developed, we have shown that PM exposure resulted in oxidative stress and DNA repair on living yeast cells. The demonstrated proof-of-concept technology here pioneers a new arena for investigating health impacts of aerosol particles in an interactive manner. Yet, its sensitivity and repeatability need to be further explored using a large set of different functional proteins.