Background Procedural pain is one of the most significant problems in neonates, especially in premature babies. Harmless and effective pain relief modalities in newborns should thus be applied. Although sucrose is the most commonly used agent, the most effective dose and concentration of sucrose is not clear. In this study, we compared the efficacy of two different doses of sucrose during venepuncture in neonates. Methods This was a prospective, randomized, double-blind study. The study was conducted during venous sampling. Oral 24% sucrose (Tool sweet((TM)) Natus Medical, San Carlos, CA, USA) was given by sterile syringe onto the anterior part of the tongue. Group 1 (n = 65) received 0.2 mL/kg 24% sucrose and group 2 (n = 64) received 0.5 mL/kg 24% sucrose. The Bernese Pain Scale for Neonates (BPSN) was used to assess the pain scores before, during and after the procedure. Results One hundred and twenty-nine premature infants were enrolled in the study, consisting of 67 girls (51.9%) and 62 boys (48.1%) aged 1-24 days (mean age, 8.34 +/- 6.25 days). There was no significant difference in BPSN score between the groups (P > 0.05). There was also no statistically significant difference in BPSN subscale scores between the groups (P > 0.05). Conclusions Twenty-four percent sucrose 0.2 mL/kg may be the minimum effective dose to relieve pain during venepuncture procedures in premature babies.