Background: Blood transfusion is a highly critical life-saving factor in neonates, especially in extremely low birth weight infants. There is a significant lack of consensus on optimal blood transfusion methods for neonates. Aim: To investigate and analyze blood transfusion practice in neonates among neonatologists and neonatal nurses in a multi-country pattern. Methods: From September 2023 to June 2024, a cross-sectional questionnaire-based study was conducted to collect data on global blood transfusion practices in neonates. A questionnaire, developed through an extensive literature review, was distributed to neonatologists and neonatal nurses primarily via e-mail, with additional distribution via social media platforms. Results: This study included a total of 180 neonatologists and neonatal nurses from 27 different countries. Almost 37.7% were working in a level 3 NICU. Approximately 37.7% of the participants stated that they transfuse blood within three hours, and approximately 45.5% stated they usually use 15 mL/kg of blood. After receiving a transfusion, 99.4% of the participants mentioned that they continue to check the vital signs. More than half (72.2%) of NICU practitioners use filters when giving blood. Regarding written instructions and guidelines in the unit for blood transfusion, the majority (84.4%) stated having them in their units, of which, 86.8% mentioned that blood transfusion threshold stated in the guidelines either using hemoglobin or hematocrit. Conclusions: This study found variability in blood transfusion practices around the world. While most have developed neonatal blood transfusion guidelines, certain countries still lack national protocols. Establishing comprehensive guidelines is essential to standardizing procedures, thereby minimizing the risk of inappropriate or unsafe blood transfusions in this neonatology practice.