Abstract: Tobacco smoke contains a large number of harmful substances and has been widely proved to be able to induce a variety of human diseases. Human eyes are exposed to ambient air for a long time in daily life and are susceptible to air pollutants such as tobacco smoke in the air. Therefore, direct monitoring of characteristic components of tobacco smoke in human tears is expected to efficiently assess the exposure level of tobacco smoke to human eyes. Due to human tears at the microliter level, it's a challenge to analyse tears by conventional sample collection and chemical analysis methods. Mass spectrometry (MS) has the unique advantages of high sensitivity, specificity and speed, especially, the development of ambient MS with solid substrates has greatly facilitated direct sample analysis with little or no sample pretreatment. Schirmer paper strip is clinically used for sampling of human tears, which is safe, non-invasive, and can be used for measuring the volume of tears. In this study, a small Schirmer paper strip was further prepared for noninvasive microsampling of human tears which exposed to tobacco smoke, and high voltage and organic solvent were applied onto Schirmer paper to directly generate paper spray ionization for ambient MS analysis. The good analytical performances, including sensitivity (LOD: 0.1 μg/L, S/N≥3; LOQ: 1.0 μg/L, S/N≥10), precision (RSD: 4.89%-7.61%, n=5, with internal standard), recovery (98%-102.1%, n=5), and linear range (0.5-50 μg/L, R²=0.991 8) were achieved. Monitoring nicotine in human tears under different exposure time was also investigated, showing that the concentration of nicotine would increase with increasing the exposure time ranging from 0 to 8 min. The nicotine could reach to 45 μg/L after burning a cigarette (about 8 min). The retention time of nicotine in human tears was found to about 20 min (less than 5%), and the half-life time was about 4 min. Overall, the results showed that Schirmer paper spray mass spectrometry can be used for qualitative and quantitative detection of trace environmental exposures such as nicotine in human eyes with many advantages, including safe, noninvasive, easy-to-operation, low-consumption as low as 1.0 μL, and thus has potential for many applications in environmental exposure and human health.