Background: Performing exercises or playing sports with an incorrect technique can cause musculoskeletal injuries. Objectives: The present study was conducted to compare the hamstring to quadriceps activation ratio when landing in a two-way spike in female volleyball players. Methods: In this cross-sectional study, 15 elite female volleyball players performed triple jump spikes (TJS) with their toe in and in a neutral ankle position. Electromyography (EMG) was used to measure the muscle activity of the biceps femoris, semitendinosus, vastus medialis and vastus lateralis in two phases before and after the initial contact. Data were analyzed using the Wilcoxon test at the significance level of P < 0.05. Results: The ratio of biceps femoris activity to vastus medialis (P = 0.04) and vastus lateralis (P = 0.04) activities in TJS with a toe-in ankle decreased significantly in the feed-back phase and the ratio of semitendinosus activity to vastus lateralis activity also decreased significantly in the feed-forward phase of TJS with a natural ankle angle (P = 0.02). Conclusions: A lower muscle activity ratio for biceps femoris to vastus lateralis and medialis in the toe-in position may lower hamstring muscle support from the anterior cruciate ligament (ACL) after landing, which might increase the risk of ACL injury when landing.