This study aimed to investigate the environmental contamination of nucleic acid at 2019 novel coronavirus (2019-nCOV) vaccination site and to evaluate the effect of improvement to the vaccination process. Nucleic acid samples were collected from the surface of the objects in 2019-nCOV vaccination point A (used between 15 November 2020 and 25 December 2020) and point B (used after 27 December 2020) in a comprehensive tertiary hospital. Samples were collected from point A before improvement to the vaccination process, and from point B (B1 and B2) after improvement to the vaccination process. The real-time fluorescence polymerase chain reaction method was used for detection. The positive rate of vaccination room was 47.06% (24/51) at point A. No positive result was found in point B1 both at working hours (0/27) and after terminal disinfection (0/27). In point B2, the positive results were found in vaccine's outer packaging and staff gloves at working hours, with a positive rate of 7.41% (2/27). The positive rate was 0 (0/27) after terminal disinfection in point B2. The nucleic acid contamination in the vaccination room of 2019-nCOV vaccine nucleic acid sampling point is serious, which can be avoided through the improvement and intervention (such as personal protection, vaccination operation and disinfection methods).