Collagen hydrolysates derived from tuna tendons have potential applications in various industries, but sterilization is crucial to ensure their safety. This study investigated the effects of ethylene oxide (EtO), beta radiation, and gamma radiation sterilization methods on the structural and functional properties of collagen hydrolysates using nuclear magnetic resonance (NMR) spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and differential scanning calorimetry (DSC). EtO sterilization caused significant physical and chemical changes in the hydrolysates, as evidenced by the altered appearance and ¹H-NMR and ¹³C-NMR spectra. In contrast, beta and gamma radiation did not induce notable changes in the physical characteristics and NMR spectra. MALDI-TOF MS analysis revealed slight alterations in the molecular weight distribution after sterilization, with beta irradiation causing a minor decrease and gamma irradiation and EtO leading to small increases. DSC analysis showed shifts in the heat absorption peaks after sterilization, indicating changes in the thermal properties. The findings suggest that while all three methods effectively sterilize collagen hydrolysates, EtO causes more significant structural modifications compared to beta and gamma radiation. This study provides valuable insights into the impact of sterilization on collagen hydrolysates, facilitating the selection of appropriate methods for specific applications.