The effects of the menstrual cycle and sex hormones on knee kinematics remain unclear. The purpose of the study was to investigate the effects of the menstrual cycle and serum sex hormone concentrations on knee kinematic parameters of the 90°cutting in female college soccer athletes. Three female college soccer teams (53 subjects) participated in the study. During the first menstrual cycle, a three-step method was used to exclude subjects with anovulatory and luteal phase-deficient (LPD) (12 subjects). The subjects' menstrual cycle was divided into the menstrual phase, late-follicular phase, ovulatory phase, and mid-luteal phase (group 1, 2, 3, 4). In each phase of the second menstrual cycle, we used a portable motion analysis system to enter the teams and tested the sex hormones concentrations and knee kinematics parameters in three universities in turn. We found that subjects had a lower maximum knee valgus in group 4 compared with other groups. This meant that subjects had a lower biomechanical risk of non-contact anterior cruciate ligament (ACL) injury in the mid-luteal phase. There was no significant correlation between serum estrogen, progesterone concentration, and knee kinematic parameters. This meant that sex hormones did not have a protective effect. Future studies need to incorporate more factors (such as neuromuscular control, etc.) to investigate.