Objective To explore the circadian rhythm disorder and brain functional connectivity （FC） changes of young female nurses on long-term night shift， and analyze the correlation between them. Methods A prospective case control design was adopted， a total of 9 female nurses on night shift duty and 9 age-matched female nurses on day shift were included as the research objects. The Pittsburgh Sleep Quality Index （PSQI） and Epworth Sleepiness Scale （ESS） were completed. The levels of serum melatonin and cortisol were collected at 5 time points （2：00， 6：00， 10：00， 16：00 and 22：00） within 24 hours， and the whole brain was scanned with resting fMRI. The hypothalamus was used as the seed area for brain FC analysis. Spearman correlation analysis was used to analyze the correlation between brain FC and serum cortisol and melatonin levels of nurses in the two groups， as well as the correlation between age， PSQI score， ESS score and working years of nurses on night shift. Results The PSQI and ESS scores of the night shift nurse were higher than those of the day shift nurse ［（8.44±3.90） vs （3.11±1.9）； 8.0（6.0， 11.0） vs 5.0（2.0， 2.5）］， and the differences were statistically significant （P＜ 0.01）. The serum melatonin level of the night shift nurse was lower than that of the day shift nurse ［（135.18±20.84） pg/ml vs （185.83±25.97） pg/ml］， the difference was statistically significant （F=69.653，P ＜ 0.01）， and the rhythm curve was disordered. The serum cortisol level of the night shift nurse was higher than that of the day shift nurse ［（7.64±0.60） nmol/L vs （6.84±0.75） nmol/L］， and the difference was statistically significant （F=211.081， P＜ 0.01）. The brain FC from hypothalamus to right cingulate gyrus， right putamen and cerebellar vermis of the night shift nurse was stronger than that of the day shift nurse， the difference was statistically significant （P＜ 0.05）. The FC enhancement from hypothalamus to cerebellar vermis of the night shift nurses was positively correlated with ESS score （r=0.501， P=0.034）. Conclusions Nurses on long-term night shift may have decreased sleep quality， decreased melatonin level at night and cortisol rhythm disorder， accompanied by resting brain FC changes.