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Background Sleep duration of the undergraduates are closely related to their health, but there is no conclusion about the best sleep duration for undergraduates. And there is a lack of research about the dose-response relationship between sleep duration and Traditional Chinese Medicine (TCM) constitution. Objective To explore the relationship between sleep duration and TCM constitution for undergraduates, and to provide reference for their health education about sleep duration. Methods This study used a combination of purposeful sampling and snowball sampling methods to conduct on-site questionnaire survey on students from 143 universities including Beijing University of Chinese Medicine, Harbin Institute of Technology (Weihai), Peking University, Northwestern Polytechnical University from January to June 2019. We used the Constitution in Chinese Medicine Questionnaire of 41-item (CCMQ-41) to assess the TCM constitution of undergraduates, and used a self-designed questionnaire to measure socio-demographic information and lifestyle behaviors. This study used the restricted cubic spline fitting multiple linear regression model to analyze the dose-response relationship between sleep duration and TCM constitution with taking sleep duration as the independent variable and TCM constitution score as the dependent variable, and controlling for related confounding variables. Results This study collected 1 003 valid questionnaires. The response rate was 98.2%. Pearson correlation analysis showed that the sleep duration was negatively correlated with the score of qi depression (r=-0.067, P=0.034). After controlling for confounders, multiple linear regression analysis showed that there was no linear relationship between sleep duration and TCM constitution scores (P>0.05). The dose-response relationship analysis showed that the highest point of gentleness score and the lowest point of biased constitutions score (except specific diathesis) had a sleep duration of about 8 h. When the sleep duration was >8.5 h, the gentleness score showed a tendency to decrease, and the scores of qi deficiency and qi depression had a tendency to increase (P<0.05) . Conclusion Considering the health status of TCM constitutions, the best daily sleep duration for undergraduates is about 8 h. The sleep duration should be restricted to be less than 8.5 h to prevent the possibility of biased constitution.