Evidence illustrated that alleviating pain in the preterm infants improve physiological, behavioral, and provide comfort. Mothers' voice recorded or breast milk odor may play a critical role as a non-pharmacological pain treatment during peripheral cannulation. Thus, the present study was aimed to investigate mothers' voice recorded, odour of breast milk on preterm babys' pain and comfort response during peripheral cannulation. A quasiexperimental design was utilized on the current study. A convenient Sample of all available preterm infants (150 preterm infants) attending the Neonatal Intensive Care Unit (NICU) at Minia University Hospital for Obstetric and Pediatrics (MUHOP) and Misr El-Hora general hospital which affiliated to ministry of health and population, (50 mothers' voice recorded, 50 breast milk odor, and 50 control groups). A structured interview questionnaire sheet included preterm infant data, Preterm Infant Pain Profile (PIPP) and Premature Infant Comfort Scale (PICS) were utilized for data collection. Results of the presents study shows significant mean difference between mothers' voice recorded, breast milk odor, and control groups regarding PIPP pain and PICS comfort assessment scores in four times (before as well as during, immediately after, and 5 minutes after peripheral cannulation) at P˂ 0.0001. The study Concluded that, mothers' voice recorded and breast milk odor groups showed a significant reduction in the total mean score of preterm infant's discomfort and pain than control group during peripheral cannulation. The Study Recommended that, new researches required to evaluate the impact of mothers' voice recorded and odour of breast milk on preterm infant pain and comfort response on large sample size.