Objective To analyze the positive duration of viral nucleic acid in severe coronavirus disease 2019 (COVID-19) patients and to explore its influencing factors, so as to understand the virus clearance state and provide evidence for treatment and management. Methods The clinical data of 41 patients with severe COVID-19 were retrospectively collected; the patients were hospitalized in Sino-French New City Branch of Tongji Hospital in Wuhan, Hubei Province from Feb. 10 to Mar. 28, 2020. The detection results of viral nucleic acid, clinical information and laboratory findings in oropharyngeal secretion were collected. The time from onset to negative of viral nucleic acid was defined as the positive duration of viral nucleic acid. Multivariate Cox regression analysis was used to explore the influencing factors affecting the positive duration of viral nucleic acid in severe COVID-19 patients. Results Among 41 patients with severe COVID-19, 20 (48.8%) were males and 21 (51.2%) were females, with a median age of 68.0 (58.5, 74.0) years. The average positive duration of viral nucleic acid was (28.98±11.71) d in the patients. Multivariate Cox regression analysis indicated that diabetes mellitus, lymphocyte count and use of high dose of glucocorticoid (total hormone≥300 mg) were associated with the positive duration of viral nucleic acid (all P<0.05), while use of low dose of glucocorticoid (total hormone<300 mg) was not related to the positive duration of viral nucleic acid (P>0.05). Conclusion Positive duration of viral nucleic acid is longer in severe COVID-19 patients. Comorbidity of diabetes mellitus, decreased lymphocyte count and use of high dose of glucocorticoid (total hormone≥300 mg) are independent risk factors for positive duration of viral nucleic acid.