Background and Aim: Pain from intravenous catheter insertion is an unpleasant and common experience in therapeutic procedures. The aim of this study was to investigate the effects of using sound frequencies on the level of pain and discomfort in the process of intravenous catheter insertion. Materials and Methods: In a triple-blind clinical trial study, 300 patients were randomly divided into ten control and intervention groups. Patients in the intervention groups received sound frequencies of 10 to 200 Hz with an intensity of 50 dB. Pain intensity, heart rate changes, number of attempts for successful sampling, and time required for the procedure were measured. Data were analyzed using Prism 10 software. Results:Pain level in the intervention groups were significantly lower with increasing sound frequency (P<0.05), while comfort level increased (P>0.05). There was no statistically significant difference in the time required to insert the intravenous catheter and the number of times required between the groups (P<0.05). In the intervention groups with increased sound frequencies, the intensity of the increase in heart rate was significantly reduced (P<0.05). Conclusion: The results of our study showed that the use of sound frequencies can effectively reduce acute pain and its hemodynamic consequences, especially at higher frequencies. It seems that sound frequencies play a role in modulating pain perception and its pathways in the central nervous system.