Abstract: Objective To analyze the positive rates of 2019-nCoV by real-time RT-PCR using various biological samples in severe/critically ill patients,and explore their clinical significance. Methods The confirmed severe/critically ill cases of COVID-19 in the fifth ward of Renmin Hospital of Wuhan University from January 31 to February 19 in 2020 were tested for 2019-nCoV by real-time fluorescence reverse transcriptase polymerase chain reaction.Positive rates of different specimens including nasopharyngeal swabs,sputum,blood,feces and anal swabs were compared. Results A total of 19 severe/critically ill COVID-19 cases were included.After two weeks' treatment,positive rate of 2019-nCoV in the nasopharyngeal swab specimens was 42.10% (8/19),and it was 68.42% (13/19) in the sputum,5.26% (1/19) in the blood,21.05% (4/19) in the feces,10.52% (2/19) in the anal swab. Conclusion Thepositive rate of 2019-nCoV nucleic acid from sputum specimens of severe/critical ill COVID-19 patients is higher than that of nasopharyngeal swabs,blood and fecal/anal swabs.The detection using multiple biological samples can increase the detection rate of 2019-nCoV infection and reduce the false negative rate in discharged cases.