Objective. To investigate the influence of long-term night shift nurses on the composition. Methods. The feces of 30 night shift nurses (test group) and 30 day shift nurses (control group) over 1 year were collected, and double-end sequencing based on bacterial 16S rDNA V3 + V4 and fungal 18S rDNA V3 + V4 was used to determine the differences by OTU clustering, diversity, flora abundance, and differential analysis. The results of 60 samples were sequenced for a total of 3, 052. There were 418 high-quality sequences, each sample produced an average of 50, 874 high-quality sequences. The OTU cluster analysis revealed that the number of OTUs was 365. The number of day-shift shift OTUs was 362. There was no significant difference (
p
<
0.05
). There was no significant statistical difference in the Chao, Ace, Shan’non, and Simpson index between the 2 groups (
P
>
0.05
). Differential analysis of gut microflora between 2 groups showed that at the phylum level, the relative abundance of firmicutes in long-term night shift nurses was higher than in day shift nurses. However, the relative abundance of bacteroidetes was lower than that of white shift nurses (all
p
<
0.05
). At the genus level, long-term night shift nurses’ Bacteroides, Faecacterium, and Bifidobacterial were lower than that of white shift nurses (
p
<
0.05
). However, the relative abundance of Prevotella and Megomonas was higher than that of white shift nurses (all
p
<
0.05
). Conclusion. There are differences between night shift nurses, which lead to a decrease in intestinal probiotic flora and increased conditional pathogenic flora. Nursing managers should improve the intestinal flora change of night shift nurses through reasonable scheduling and dietary regulation.