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Since the 2019 COVID-19 virus outbreak, real-Time RT-PCR become the gold standard detection for COVID-19. We aimed to analyze the RT-PCR technicians’ knowledge, attitudes, and practices regarding to recent advances in RT-PCR tests for COVID-19. This is a cross-sectional study, that was conducted from 10 Nov 2022 to 20 Nov 2022, and included a total of 531 participants from China. To measure the difference in the mean scores, and analyze the factors related to knowledge, attitude, and practice toward RT-PCR by using univariate, non-parametric, and regression analysis. Of this survey, the mean knowledge, attitude, and practice scores about recent advances in RT-PCR tests for the diagnosis of COVID-19 were 13.23 (SD = 0.85, range: 13–16), 13.16 (SD = 1.71, range: 5–12), and 6.14 (SD = 1.85, range: 1–8), respectively, indicated good KAP. However, some significant predictors including education level and work status, influence knowledge. Age, marital status, and yearly income influence attitudes and practices. Our investigation can further understand the characteristics of RT-PCR technicians for the diagnosis of COVID-19, and be targeted to improve their overall ability.