Background <p>This study aimed to determine the risk of anterior cruciate ligament (ACL) injury in female college students according to sleep quality and use this information to prevent injury.</p> Methods <p>Twenty-six female university students were recruited for the present study. Participants underwent assessments for sleep quality, landing error scoring system (LESS), and electromyographic (EMG) activity for imbalances by comparing the muscle activity of the left and right rectus femoris (RF-to-RF) and biceps femoris (BF-to-BF) muscles.</p> Results <p>The good sleep group exhibited significantly lower LESS scores (good sleep group: 5.13 ± 1.46, poor sleep group: 7.09 ± 1.38, <i>p</i> = 0.004). Additionally, the EMG activity of the RF-to-RF (good sleep group: 20.82 ± 20.29, poor sleep group: 41.74 ± 24.76, <i>p</i> = 0.019) and the EMG activity of the BF-to-BF (good sleep group: 27.09 ± 25.40, poor sleep group: 109.92 ± 93.43, <i>p</i> = 0.001) were significantly lower in the good sleep group. In all participants, a positive correlation was found between the PSQI total score and EMG activity of BF-to-BF (<i>r</i> = 0.446, <i>p</i> &lt; 0.05), as well as the LESS score and EMG activity of BF-to-BF (<i>r</i> = 0.601, <i>p</i> &lt; 0.01).</p> Conclusion <p>This study demonstrated the relationship between sleep quality and ACL injury risk factors among female university students. We strongly recommend that these students improve their sleep quality to reduce and prevent ACL risk.</p>