Aim: This study aimed to understand the impact of perceived stress on the subjective happiness of Chinese healthcare workers (HCWs) and to further explore the chain-mediating role of family companionship and mental health. Background: In the face of tense doctor-patient relationships; a heavy workload; long working hours; seemingly endless shifts; potential professional title promotions; work performance assessments; and the difficult balance between family, work, and other aspects of life, HCWs are often under great pressure, which can endanger mental health and reduce subjective happiness. However, the role of healthcare workers' active participation in family companionship in mental health and subjective happiness is not clear. Method: We used a mixed research design to collect data in two locations (Hospital A and Hospital B) in Wuhan, China. A self-distributed questionnaire was assigned to HCWs through the Research Electronic Data Capture survey. A total of 368 valid surveys were obtained. Results: Hospital A's perceived stress level and mental health problems were more severe, while Hospital B had a higher subjective happiness score and more time to spend with their families. Subjective happiness was affected by children, education, occupation, health status, commuting time, and the scores of perceived stress and depression. The scores of perceived stress and mental health were significantly negatively associated with subjective happiness and family companionship, and there was a significant positive correlation between subjective happiness and family companionship. The results also showed that family companionship and mental health acted as serial mediators between perceived stress and subjective happiness. However, family companionship did not play a mediating role between perceived stress and subjective happiness. Most HCWs had work-family conflicts, and a high amount of work pressure and feelings of powerlessness and not having sufficient time were common when they accompanied their families. Conclusions: HCWs had a high level of perceived stress and psychological distress, and their subjective happiness score was lower than that of the general population. Many HCWs experienced negative emotions when taking care of their families. Only a small number of people had enough time to spend time with their families and perform more prominently in busier hospitals. More importantly, perceived stress can indirectly have an impact on subjective happiness through a chain-mediating effect of family companionship and mental health, and family companionship may not always promote subjective happiness unless mental health is maintained. Therefore, in the future, we can consider carrying out interventions based on family companionship and mental health among HCWs to promote the healthy and harmonious development of individuals, families, and hospitals.