Objective To assess the status of character strengths, strengths use and subjective well-being among male nurses, and to analyze their relationship. Methods A total of 744 registered male nurses from tertiary and secondary general hospitals and specialized hospitals in Anhui province were selected using convenient sampling, then they were investigated utilizing Three-Dimensional Inventory of Character Strengths (TICS), Strengths Use Scale (SUS) and General Well-Being Schedule (GWB). Results The average score of TICS and SUS among the male nurses was (3.53±0.58) and (4.61±1.16) respectively, and the total GWB score was (72.89±12.73). Character strengths score was positively correlated with the score of strengths use and subjective well-being, and strengths use score had a positive correlation with subjective well-being score (P<0.01 for all). The score of character strengths had a direct positive predictive effect on the score of subjective well-being, and strengths use was a mediator between character strengths and subjective well-being, the intermediary effect was 0.101, which accounted for 18.70% of the total effect. Conclusion Male nurses′ character strengths and strengths use are closely related to subjective well-being, and character strengths can indirectly affect subjective well-being through strengths use. Clinical managers should pay attention to identifying male nurses′character strengths, guide them to correctly apply their strengths, so as to improve their subjective well-being.