BACKGROUND: Working in shifts can disrupt circadian rhythm and reduce sleep duration, which have a detrimental effect on cognitive function. Shift work is often a special requirement for nurses to provide continuous service for patients. The Stroop task is a classic method of executive function (EF) applied in neuroimaging researches. Functional near-infrared spectroscopy (fNIRS) has created new opportunities for investigating the hemodynamics of cerebral activated regions during executive function. However, there has been no study exploring cerebral hemodynamics changes related to shift work by fNIRS in nurses during performing a Stroop paradigm. The purpose of our study was to investigate the effects of shift work on fNIRS-based cerebral functions during the Stroop task among nurses in a real clinical environment. METHODS: Nurses registered in the Department of Neurology were eligible and consecutively included if they were simultaneously responsible for the day, evening and night shifts on the shift work schedule. A multi-channel fNIRS imaging system (NirScan, Danyang Huichuang Medical Equipment Co. Ltd, China) was used to acquire each participant's cerebral hemodynamic activities during performance of the Stroop task, which was performed for each subject separately before and after three working shifts. RESULTS: Eighteen nurses with certification were included in our study. Cerebral cortical activation and functional connectivity were significantly changed during Stroop test after day-shift (all p < 0.05, FDR corrected), indicating a leading role of left PFC. Further, we identified the reaction time under incongruent task before day shift, was positively correlated with LPFC (r = 0.507, p = 0.038), RPFC (r = 0.547, p = 0.023) and BPFC activation (r = 0.512, p = 0.036). CONCLUSIONS: Our study further supported fNIRS as a useful functional imaging technique for monitoring brain activity in healthcare providers. Also, brain activation and functional connectivity during Stroop task consistently showed that working in day shift was related to decreased brain hemodynamic activities in nurses. Further, our findings would be helpful for leadership in clinical management on decision-making about arrangements for shift work.