This study presents the detection of a high-frequency quasi-periodic oscillation (QPO) in the Seyfert galaxy NGC 1365 based on observational data obtained by XMM-Newton in January 2004. Utilizing the weighted wavelet Z-transform (WWZ) and Lomb-Scargle periodogram (LSP) methods, a QPO signal is identified at a frequency of 2.19 × 10−4 Hz (4566 s), with a confidence level of 3.6σ. The signal is notably absent in the lower 0.2–1.0 keV energy band, with the primary contribution emerging from the 2.0–10.0 keV band, where the confidence level reaches 3.9σ. Spectral analysis shows that there are multiple absorption and emission lines in the high-energy band (> 6 keV). The correlation between the QPO frequency (fQPO) and the mass of the central black hole (MBH) of NGC 1365 aligns with the established logarithmic trend observed across black holes, indicating the QPO is of high frequency. This discovery provides new clues for studying the generation mechanism of QPOs in Seyfert galaxies, which helps us understand the accretion process around supermassive black holes and the characteristics of strong gravitational fields in active galactic nuclei.