Neoseiulus bicaudus (Wainstein) (Acari: Phytoseiidae) is a generalist predatory mite that consumes several pest species, including Tetranychus turkestani (Ugarov et Nikolskii) (Acari: Tetranychidae) in the Xinjiang Uygur Autonomous Region. The release numbers of predatory mites are based on the populations of target pests and their ability to control them. Populations of T. turkestani and T. truncatus Ehara (Acari: Tetranychidae) often coexist and damage many crops. To determine whether the presence of the non-target prey T. truncatus affects the ability of N. bicaudus to control the target prey T. turkestani. The study evaluated the predation rate and functional response of N. bicaudus to 4 stages of T. turkestani in the presence of T. truncatus. The consumption of T. turkestani by N. bicaudus gradually decreased as the proportion of T. truncatus increased. The functional response of N. bicaudus to T. turkestani was not changed when T. truncatus was presented, which was consistent with a type II response. The attack rate of N. bicaudus on the egg, larva, and nymph of T. turkestani was significantly decreased and the handling time of N. bicaudus on T. turkestani was significantly extended when T. truncatus was presented. The preference index showed that the preference of N. bicaudus for eggs and female adults of T. turkestani decreased with increasing density of T. turkestani in the same proportion as T. truncatus. The presence of T. truncatus can negatively affect the predation of T. turkestani by N. bicaudus. We suggest that the number of N. bicaudus released to control T. turkestani should be increased when T. truncatus coexist.