Determining the presence and severity of knee osteoarthritis (OA) is a valuable application of inertial measurement units (IMUs) in the remote monitoring of patients. This study aimed to employ the Fourier representation of IMU signals to differentiate between individuals with and without knee OA. We included 27 patients with unilateral knee osteoarthritis (15 females) and 18 healthy controls (11 females). Gait acceleration signals were recorded during overground walking. We obtained the frequency features of the signals using the Fourier transform. The logistic LASSO regression was employed on the frequency domain features as well as the participant’s age, sex, and BMI to distinguish between the acceleration data from individuals with and without knee OA. The model’s accuracy was estimated by 10-fold cross-validation. The frequency contents of the signals were different between the two groups. The average accuracy of the classification model using the frequency features was 0.91 ± 0.01. The distribution of the selected features in the final model differed between patients with different severity of knee OA. In this study, we demonstrated that using logistic LASSO regression on the Fourier representation of acceleration signals can accurately determine the presence of knee OA.