Abstract This study evaluated retinal and choroidal microvascular changes in night shift medical workers and its correlation with melatonin level. Night shift medical workers (group A, 25 workers) and non-night shift workers (group B, 25 workers) were recruited. The images of macula and optic nerve head were obtained by swept-source OCT-angiography. Vessel density of retina, choriocapillaris (CC), choriocapillaris flow deficit (CC FD), choroidal thickness (CT) and choroidal vascularity index (CVI) were measured. 6-sulfatoxymelatonin concentration was analyzed from the morning urine. CC FD and CVI were significantly decreased and CT was significantly increased in group A (all P < 0.05). 6-sulfatoxymelatonin concentration was significantly lower in group A ( P < 0.05), which was significantly positively correlated with CC FD size ( r = 0.318, P = 0.024) and CVI of the most regions (maximum r -value was 0.482, P < 0.001), and was significantly negatively associated with CT of all regions (maximum r -value was − 0.477, P < 0.001). In night shift medical workers, the reduction of melatonin was significantly correlated with CT thickening, CVI reduction and CC FD reduction, which suggested that they might have a higher risk of eye diseases. CC FD could be a sensitive and accurate indicator to reflect CC perfusion.