We aimed to explore the association between family conflict and HPA axis activity, especially with respect to the potential modulating effect of puberty. A total of 205 adolescents and 244 adult parents were recruited. Family conflict was assessed by the family conflict subscale of the Family Environmental Scale and serial salivary cortisol was measured in all participants. A marginally lower AUCg at 30 minutes after wake up in the morning and a significant lower AUCg at 60 minutes and 90 minutes in adult parents with high family conflict was found when compared to those with low family conflict. In adolescents, there were significant interaction effects between pubertal status and family conflict on AUCg (interaction p values <0.05). Among the adolescents with low family conflict, those at late/post pubertal status had higher AUCg than their pre/early pubertal counterparts but this difference was not observed in the adolescents with high family conflict. Adverse family environment is associated with HPA axis dysfunction in adults and late/post pubertal adolescents and pubertal maturation plays a critical role in modulating the association between family environment and HPA axis function.