Cigarette smoking is believed to affect thyroid function and autoimmune thyroid disease. However, there is little information to analyze their association using objective biomarkers. The aim of this study was to investigate the dose-related effect of smoking on thyroid hormones and thyroid peroxidase antibody (TPO Ab) using urinary cotinine levels and a population-based cohort. The present study used the thyroid hormone and urinary cotinine dataset from sixth Korean National Health and Nutrition Examination Survey in 2014 and 2015, and a total of 4249 participants were included. Dose-response relationships between thyroid hormone (free T4, TSH, and TPO Ab) and urinary cotinine levels were estimated using ANCOVA after adjustment for all variables (age, height, weight, alcohol, exercise, and log- transformed iodine in urine). Urinary cotinine level was negatively correlated with TSH. The estimated coefficients were −0.0711 in males and −0.0941 in females (p < 0.0001). TPO Ab titer was positively correlated with cotinine levels in males (p < 0.0001). Our findings indicated a significant dose-related effect of urinary cotinine level on thyroid function, and thyroid autoimmunity.