Background With the spread of Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection, its effect on society is amplified. We aimed to describe the viral detection results across different timepoints throughout the disease course. Methods A retrospective study of 301 confirmed COVID-19 patients hospitalized at Tongji Hospital in Wuhan, China, were included. Demographic characteristics of the patients were collected. Upper respiratory specimens (throat and/or nasal swabs) were obtained and analyzed by real-time RT-PCR for SARS-CoV-2 infection. Period of viral infection and the contagious stage were analyzed. Results Of 301 hospitalized COVID-19 patients, the median age was 58 years and 51.2 % were male. The median period between symptoms presence and positive SARS-CoV-2 RT-PCR results was 16 days (IQR, 10–23, N = 301). The median period between symptoms presence and an effective negative SARS-CoV-2 RT-PCR result was 20 days (IQR, 17–24; N = 216). Infected patient ≥65 years old stayed contagious longer (22 days vs 19 days, p = 0.015). Although two consecutive negative results were confirmed in 70 patients, 30 % of them had positive viral test results for the third time. Using specimens from nasal swabs to run the RT-PCR test showed a higher positive rate than using specimens from throat swabs. Conclusions This large-scale investigation with 1113 RT-PCR test results from 301 COVID-19 patients showed that the average contagious period of SARS-CoV-2 infected patients was 20 days. Longer observation period and more than 2 series of negative viral test are necessary for patients ≥65 years.