Background: In the present study, we investigated the association between substance use by adolescents and parental smoking status based on data from the 2016 Korean Youth Risk Behavior Web-based Survey, a national school-based survey. Methods: Data from a nationally representative sample of Korean adolescents aged 12–18 years (n = 65,528) were analyzed, and the risk of substance use according to the parental smoking status was investigated. Results: We found that smoking by both parents was a greater risk factor for substance use by adolescents than smoking by any one parent. Moreover, maternal smoking was a greater risk for substance use by adolescents than paternal smoking. We also investigated sex differences in the risk of substance use as a result of the parental smoking status. The differences in the substance use status according to the sex of both parents and children were also identified after adjustment for second-hand smoking. Conclusions: Accurate evaluation of the family smoking environment and whole-family interventions are necessary for preventing and intervening in substance use by adolescents.