Objectives The objectives of this study are to determine parental and children's hair nicotine levels, their relationships as well as to investigate the association of smoking status of the fathers with mothers' and children's hair nicotine. Methods A cross-sectional study design was conducted among 124 families who were participants of the Universiti Sains Malaysia Pregnancy Cohort Study. Both parents with their 2 years old children joined this study. A total of 92 hair samples of fathers, 124 hair samples of mothers and 111 hair samples of children were collected and analyzed by gas chromatography-mass spectrometry. Results Of total, 52.4 % of the fathers reported smoking. None of the mothers were smokers. Hair nicotine levels of fathers were found to be significantly correlated with mothers (r = 0.233, p = 0.026) and children (r = 0.508, p \ 0.001). Children living with smoking fathers had significantly higher median hair nicotine level compared to the children of non-smoking fathers (6.08 vs 0.22 ng/mg, p = 0.046). However, this association was not seen in the mothers. Quantile regression showed significant association between fathers' and children's hair nicotine. Conclusion There is a positive relationship between fathers' hair nicotine with mother's and children's hair nicotine. Living with smoking fathers can contribute to higher hair nicotine levels in children but not in mothers.