Studies have used 8-hydroxydeoxyguanosine (8-OHdG) as a biomarker to detect systemic oxidative DNA damage associated with oxidative stress. However, studies on the association between exposure to tobacco smoking and urinary 8-OHdgG give inconsistent results. Limited studies have estimated the oxidative stress among office workers. This study assessed the association between urinary 8-OHdG and cotinine for office workers. Workers (389) including smokers, ex-smokers and non-smokers from 87 offices at high-rise buildings in Taipei participated in this study with informed consent. Each participant completed a questionnaire and provided a spot urine specimen at the end of work day for measuring urinary 8-OHdG and cotinine. The carbon dioxide (CO2) levels in workers' offices were also measured. The questionnaire reported socio-demographic characteristics, life styles and allergic history. The urinary 8-OHdG level increased with the cotinine level among participants (Spearmans' rho = 0.543, p < 0.001). The mean of urinary 8-OHdG and cotinine was 5.81 ± 3.53 μg/g creatinine and 3.76 ± 4.06 μg/g creatinine, respectively. Comparing with non-smokers, the adjusted odds ratio (OR) of having urinary 8-OHdG greater than the median level of 4.99 μg/g creatinine was 5.30 (95% confidence intervals (CI) = 1.30-21.5) for current smokers and 0.91 (95% CI = 0.34-2.43) for former smokers. We also found workers exposed to 1,000 ppm of CO2 at offices had an adjusted OR of 4.28 (95% CI = 1.12-16.4) to have urinary 8-OHdG greater than 4.99 μg/g creatinine, compared to those exposed to indoor CO2 under 600 ppm. In conclusion, urinary 8-OHdG could represent a suitable marker for measuring smoking and CO2 exposure for office workers.