The mercury removal capacity of biochar can be improved by plasma modification and the functional group is an important factor affecting the mercury removal. This paper examines factors such as discharge voltages, gas flow rates, chlorine concentrations and discharge times that may affect functional groups on the surface of biochar. The mercury removal performance of the tobacco stem biochar prepared under different Cl-2 plasma modification conditions was investigated using a fixed bed reactor. The number of C-Cl bonds and carboxyl increased after modification and decreased in mercury removal. Longer discharge times can destroy the biochar surface and possibly cause a decrease in the number of active sites. Increasing the discharge voltage promotes the formation of C-Cl bonds and carboxyl groups. Excessive gas flow rates cause active chlorine to be carried out of the reactor quickly which reduces the formation of C-Cl. The amount of C-Cl bonds increases with increased chlorine concentration. In the modified biochar, C-Cl and carboxyl take part in the adsorption of mercury to form HgCl2 and HgO, while the unmodified biochar is mainly physically adsorbed Hg-0. C-Cl is the main functional group participated in mercury removal and the carboxyl is a secondary functional group. The proportion of HgCl2 is at least 68.2% in the used modified biochar. Under optimum conditions, the initial mercury removal efficiency of modified tobacco biochar was 99.1%, and the efficiency was still 79.1% after 100 min.