Objective: To explore the association of vitamin D deficiency with anxiety and depressive symptoms as well as their comorbidity, so as to provide references for improving the mental health of children and adolescents. Methods: From October to December 2021, a total of 1 323 students from four middle schools in Shenzhen were selected by using convenience sampling method for a questionnaire survey and physical examination. Fasting blood samples were collected to detect vitamin D levels. The Chi square test and multiple Logistic regression model were used to analyze the association of vitamin D deficiency with anxiety and depressive symptoms and their comorbidity among middle school students. Results: The detection rates of anxiety, depressive, and thier comorbidities in middle school students were 33.0%, 21.5% and 16.3%, respectively. After adjusting for confounding factors, vitamin D deficiency was positively associated with the risk of anxiety symptoms ( OR=1.59, 95%CI =1.02-2.46), depressive symptoms ( OR=1.94, 95%CI =1.22-3.09) and anxiety depressive comorbid symptoms ( OR=1.75, 95%CI =1.06-2.90) in secondary school students ( P ＜0.05). Conclusion Vitamin D deficiency is associated with an increased risk of anxiety, depressive and their co morbidity symptoms in middle school students, and the directionality of the association should be further studied.