AimTo investigate the alterations of functional brain activity and connectivity in female nurses working on long‐term shifts and explore their correlations with work‐related psychological traits.DesignAn exploratory cross‐sectional study.MethodsThirty‐five female nurses working on long‐term shifts (shift nurses) and 35 female nurses working on fixed days (fixed nurses) were enrolled. After assessing the work‐related psychological traits, including burnout, perceived stress, anxiety, and depression of nurses, the fractional amplitude of low‐frequency fluctuations (fALFF) and region of interest (ROI)‐based functional connectivity (FC) analyses were performed to investigate the differences of brain spontaneous activity and functional connectivity between these two groups of nurses. Thereafter, correlations between the functional brain parameters (fALFF and FC) and clinical metrics were investigated among the shift nurses.ResultsCompared to fixed nurses, shift nurses had higher burnout, perceived stress and depression scores, lower fALFF in the right dorsolateral prefrontal cortex (dlPFC), left and right superior parietal lobule (SPL), bilateral anterior cingulate cortex (ACC), and higher fALFF in the right superior/middle temporal gyrus, as well as decreased FC between the right dlPFC (the selected ROI) and bilateral ACC, left and right inferior frontal/orbitofrontal gyrus (IFG/IOFG), right SPL, and left middle occipital gyrus (voxel‐level p < 0.001, cluster level p < 0.05, GRF correction). Correlation analyses demonstrated that the fALFF value of the right dlPFC was significantly correlated with the burnout and anxiety scores, the FC value of the right dlPFC‐right SPL was correlated with the perceived stress and burnout scores, the FC value of the right dlPFC‐right IFG/IOFG was correlated with the burnout score in shift nurses (p < 0.05).ConclusionShift nurses had work‐related altered functional activity and connectivity in the right frontoparietal network, which provided objective and visualised evidence to clarify the hazards of long‐term shift work on female nurses.Patient or Public ContributionSeventy nurses participated deeply as subjects in this study. These findings are expected to draw managers' attention to the harmful influences of shift work on nurses.