Objective To explore the suspected secondary SARS-CoV-2 infection caused by nucleic acid sampling activities during the epidemic of SARS-CoV-2 infection. Methods A local epidemic of SARS-CoV-2 Delta variant infection broke out in a city in Jiangsu Province from July to August 2021. Based on the detection information in the SARS-CoV-2 nucleic acid testing information system of Jiangsu Province in this period, combined with the epidemiological history and laboratory genetic sequencing results of the patients, suspected cases of secondary infection caused by sampling activities were screened, and the time interval between onset/first positive test of associated cases and single exposure to secondary cases was calculated. Risks of secondary infection under different sampling modes at sampling locations were assessed. Results A total of 8 sampling locations were suspected of secondary infection, involving 42 cases, among which 32 cases had participated in large-scale nucleic acid sampling activities in village-L. Among the 42 cases of secondary SARS-CoV-2 infections, 25 had a clear infection source. Their single exposure with associated cases occurred 3 and 7 days before the onset of associated cases, 0, 1, 3, 4 days before and 1 day after the first positive test of associated cases, respectively. The majority of the single exposure occurred 1 day after the first positive test of associated cases, accounting for 64.0%(16/25). The risk of secondary infection at sampling locations under different sampling modes was: concentrated sampling of large-scale population >sampling based on streets or communities > door-to-door sampling. Conclusion The risk of secondary SARS-CoV-2 infection at centralized sampling location of large-scale population is relatively high, which increases the risk of subsequent transmission and spread of the epidemic. The sampling modes based on streets or communities and door-to-door sampling are preferred, but maintaining order at the sampling location and personal protection are still key measures to avoid secondary SARS-CoV-2 infection at the sampling location.