The majority of shift nurses are female, there is still an expectation that they fulfil the traditional role of women in the family in Asia, often conflicting with shift work, increases stress, and affects cortisol secretion patterns. This study was to understand the changes in the cortisol awakening response (CAR) and work stress in nursing personnel working in different shifts. We recruited 41 female shift nurses. We administered the Taiwan Nurse Stress Checklist (NSC), and the nurses themselves collected saliva samples upon waking and 30 minutes after waking for three consecutive days at home. The saliva samples enabled us to analyze the increase in cortisol levels following waking (CARi) of nurses working different shifts (day, evening, and night). We then analyzed the data obtained using a hierarchical linear model (HLM). The results indicated that in terms of stress from the inability to complete personal tasks, the regression coefficients of night-shift nurses vs. day-shift nurses (B = 4.39, p < .001) and night-shift nurses vs. evening-shift nurses (B = 3.95, p < .001) were positive, which means that night-shift nurses were under significantly greater stress than day-shift and evening-shift nurses. With regard to CARi, the regression coefficients of night-shift nurses vs. day-shift nurses (B = −3.41, p < .001) and night-shift nurses vs. evening-shift nurses (B = −2.92, p < .01) were negative, which means that night-shift nurses have significantly lower CARi values than day-shift and evening-shift nurses. With regard to cortisol levels 30 minutes after waking, the regression coefficients of night-shift nurses vs. day-shift nurses (B = −3.88, p < .01) and night-shift nurses vs. evening-shift nurses (B = −3.31, p < .01) were negative, which means that night-shift nurses have significantly lower cortisol levels 30 minutes after waking than day-shift and evening-shift nurses. These results indicate that female night-shift nurses display the lowest CARi and cortisol levels 30 minutes after waking and are more negatively affected by being unable to complete personal tasks.