Aim: Explore the optimal cut-off values for plasma nicotine and its metabolites in assessing smoking status and quantify the association between individual tobacco exposure and pulmonary nodules (PNs).Materials & methods: A total of 2245 plasma samples were included for the determination of nicotine (Nic), cotinine (Cot) and trans-3'-hydroxycotinine (OHCot) concentrations. The receiver operating characteristic curve was used to determine the optimal biomarkers reflecting smoking status. Binary logistic regression, restricted cubic spline and generalized linear model were used to analyze the association of nicotine and its metabolites with PNs. Quantile g-computation was used to investigate the mixed effects between them.Results: Cot was found to be the best biomarker of self-reported active-passive smoking, with optimal thresholds of 9.06 and 1.26 ng/ml, respectively. Except for OHCot, increased concentrations of Cot, Nic, total nicotine equivalent (TNE2) and TNE3 were significantly positively associated with the risk of PNs, whereas nicotine metabolite ratio presented a negative association. The mixed effects of OHCot, Cot and Nic were associated with PNs, with an odds ratio of 1.17 and a 95% CI of 1.05-1.30.Conclusion: Nicotine and its metabolites as potential biomarkers of tobacco exposure were significantly associated with PNs.