Background Due to the COVID-19 outbreak, all teaching activities in nursing schools were suspended in China, and many nursing students were summoned to work in hospitals to compensate for the shortage of manpower. This study examined the prevalence of fatigue and its association with quality of life (QOL) among nursing students during the post-COVID-19 era in China. Methods This was a multicenter, cross-sectional study. Nursing students in five Chinese universities were invited to participate. Fatigue, depressive and anxiety symptoms, pain and QOL were measured using standardized instruments. Results A total of 1,070 nursing students participated. The prevalence of fatigue was 67.3% (95% CI [64.4–70.0]). Multiple logistic regression analysis revealed that male gender (P = 0.003, OR = 1.73, 95% CI [1.20–2.49]), and being a senior nursing student (second year: OR = 2.20, 95% CI [1.46–3.33], P < 0.001; third year: OR = 3.53, 95% CI [2.31–5.41], P < 0.001; and fourth year OR = 3.59, 95% CI [2.39–5.40], P < 0.001) were significantly associated with more severe fatigue. In addition, moderate economic loss during the COVID-19 pandemic (OR = 1.48, 95% CI [1.08–3.33], P < 0.015; compared to low loss), participants with more severe depressive (OR = 1.48, 95% CI [1.22–1.78], P < 0.001) and anxiety symptoms (OR = 1.12, 95% CI [1.05–1.20], P = 0.001), and more severe pain (OR = 1.67, 95%CI [1.46–1.91], P < 0.001) were significantly associated with reported more severe fatigue. After controlling for covariates, nursing students with fatigue had a lower overall QOL score compared to those without (F_{(1, 1070)} = 31.4, P < 0.001). Conclusion Fatigue was common among nursing students in the post-COVID-19 era. Considering the negative impact of fatigue on QOL and daily functioning, routine physical and mental health screening should be conducted for nursing students. Effective stress-reduction measures should be enforced to assist this subpopulation to combat fatigue and restore optimal health.