This study aimed to investigate the effects of knee flexion during the preparation phase of a serve on the tennis serve performance, using inertial sensors. Thirty-two junior tennis players were divided into two groups based on their maximum knee flexion during the preparation phase of serve: Smaller (SKF) and Greater (GKF) Knee Flexion. Their racket velocity, racket height, and knee extension velocity were compared during the tennis serve. Inertial sensors tracked participants’ shank, thigh, and racket motions while performing five first, flat, and valid serves. Knee flexion was analysed during the preparation phase of serve, knee extension velocity after this phase, racket velocity just before ball impact, and racket height at impact. Pre-impact racket velocity (mean difference [MD] = 3.33 km/h, p = 0.004) and the knee extension velocity (MD = 130.30 °/s, p = 0.012) were higher in the GKF than SKF; however, racket impact height was not different between groups (p = 0.236). This study’s findings support the importance of larger knee flexion during the preparation phase of serve-to-serve performance. This motion should be seen as a contributor to racket velocity.