Background The hamstring exercises are recommended to prevent and rehabilitate hamstring strain injury. A single-leg deadlift is a hamstring exercise, which is used in the early and late stages of rehabilitation. In the single-leg deadlift, the muscle activity depends on the forward trunk lean angle, and it is necessary to study which muscles are used dominantly depending on the angle. Objectives This study aimed to compare the effect on the biceps femoris(BF) and semitendinosus(ST), which is a hamstring, muscle activity during single-leg deadlift according to different forward trunk lean angles. Methods Seventeen female adults participated in this study. The subjects were trained to familiarize themself with the measurement process in advance for 10 minutes before measuring. During single-leg deadlift according to 30°, 60°, and 90° of forward trunk lean angles, the BF and ST muscles activities were measured through electromyography (EMG). Data were analyzed by one-way repeated-measures analysis of variance, and a Bonferroni adjustment was performed if a significant difference was found. Statistical significance levels were set at α = 0.05. Results Muscles activities of the BF and ST have significantly differed among three conditions (30°, 60°, and 90° of forward trunk lean). In addition, there was a significant difference between 30° and 90° and 60° and 90°. Both muscle activities were significantly highest at 90° of forward trunk lean. Conclusions Our results indicate that 90° of forward trunk lean was more effective in the hamstring muscle activity than at other angles. These findings support that a single-leg deadlift at 90° of forward trunk lean is a proper hamstring muscle-strengthening exercise.