Abstract: Tuberculosis (TB) is a serious infectious disease caused by Mycobacterium. tuberculosis. In recent years, with the emergence of drug-resistant forms, the development of new anti-tuberculosis drugs is urgently needed. In this study, we used Mycobacterium marinum (M. marinum), which is highly similar to M. tuberculosis, to establish a M. marinum infected-zebrafish model and quantitative PCR (qPCR) method for bacterial count analysis. The results showed that injecting M. marinum into the yolk sac is an efficient and convenient way to infect zebrafish embryos. By counting the survival rate of infected zebrafish and the number of bacteria in zebrafish by ZiehlNeelsen staining, we analyzed the efficacy of isoniazid and rifampicin as anti-tuberculosis drugs and the synergistic effect of drugs. The results suggested that three evaluation methods exhibit good consistency. This study demon strated that zebrafish-M. marinum infection model combined with qPCR analysis is a simple and efficient method for in vivo screening and evaluation of anti-tuberculosis drugs. Animal experiments were carried out in accordance with the provisions for animal ethics in the Regulations on Laboratory Animals of Institute of Medicinal Biotech nology, Chinese Academy of Medical Sciences.