To analyze the application effect of the white noise combined with the comfort pacifier intervention for neonates during their heel blood collection. Methods: 106 neonates born in the hospital who wanted screening by the heel blood collection were selected and were divided into control group (neonates with the routine intervention during heel blood collection) and intervention group (53 neonates with the white noise combined with the comfort pacifier intervention) by means of simple random sampling method from December 2020 to December 2023. The blood collection status, and the physiological indexes and the pain response before and after blood collection of the neonates were observed and compared between the two groups. The neonatal puncture adverse events in the two groups were recorded. Results: The diameter of the blood drop (11.04±1.69mm) of the neonates in intervention group was significantly higher than that (7.86±1.03mm) of the neonates in the control group. The bleeding time (2.55±0.40s) and the crying time (44.16±5.21s) of the neonates in intervention group were significantly shorter than those (3.64±0.43s and 92.08±10.37s) of the neonates in the control group. After blood collection, the heart rate and the respiratory rate of the neonates in the two groups had increased significantly, but both of which (137.1±16.1 times/min and 48.0±5.1 times/min) of the neonates in the intervention group were significantly lower than those (153.5±16.2 times/min and 56.2±6.1 times/min) of the neonates in the control group. The SaO2 value of the neonates in the two groups after blood collection had decreased significantly, and which (92.3±2.7%) of the neonates in the intervention group was significantly higher than that (91.3±2.5%) of the neonates in the control group. The degree of pain of the neonates in the intervention group was significantly lower than that of the neonates in the control group, and the total incidence (17.0%) of adverse puncture events, such as hematoma, bleeding and ecchymosis, of the neonates in the intervention group was significantly lower than that (35.9%) of the neonates in the control group (all P<0.05). Conclusion: The white noise combined with the comfort pacifier intervention for the neonates during their heel blood collection can achieve the better effect, and which can relieve the neonatal pain and reduce the neonatal risk of puncture adverse events.