As contemporary society has become more complicated, specialized, and segmented, people are experiencing more diverse types of stress. In particular, while several factors associated with job stress have been examined among nurses, who belong to a professional group, the existing research has made no quantitative assessments of stress that reflect temporal differences in individuals. Therefore, the aim of this study is to understand the effects of job stress on alpha-amylase with regard to the working hours of nurses, to assess the variations in jobs stress over time, and provide basic data to improve the quality of nursing services. Ninety nurses working in three shifts in general, emergency, and intensive care wards of a university hospital in D City participated in this study. Salivary alpha-amylase (SAA) was extracted and analyzed at two-hour intervals from 07:00 to 15:00 from nurses on the day shift and from 23:00 to 07:00 from those working the night shift. The SAA level was highest between 23:00 and 01:00 for nurses in general wards (mean±S.D. 39.00±14.88) and between 11:00 and 13:00 for those in both intensive care units and emergency wards (mean ± S.D. 67.50 ± 62.93 and mean ± S.D. 39.67±35.96, respectively). The characteristic variation in SAA was significant between 23:00 and 01:00 (p < 0.01) and for those in their fifties or older (p < 0.01). The activation ratio of alpha-amylase, a stress reactant, showed an increase when the sympathetic nervous system was activated by mental stress; in addition, job stress was manifested with the effect of awakening at different time segments and at different ages among the nurses. With the aim of raising the level of service based on the nurses maintaining their mental health, it is necessary to focus sharply on the time segment for critical control and to conduct repetitive studies to determine the divisions of eustress critical values as well as to expand the population.