Aims/Background Establishing an intraosseous infusion (IO) pathway can rapidly open an urgent route of drug administration for critically ill patients. This study aims to assess different puncture sites on the efficacy of manual intraosseous infusion. Methods Upon applying computed tomography (CT), we compared compact bone thickness and CT values at the same individual's proximal humerus and proximal tibia puncture sites (n = 40). Additionally, cadaveric experiments were used to compare the efficiency of manual puncture at two different insertion sites of the proximal humerus and proximal tibia in the same individual (n = 5). Results The compact bone thickness and CT values at the proximal humerus were significantly lower than those at the proximal tibia. The cadaveric experiments further confirmed that the proximal humerus was superior to the proximal tibia as an insertion site, indicating the proximal humerus is a more suitable insertion site for manual bone marrow puncture needles. Conclusion Selection of the puncture site markedly influences the effectiveness of manual intraosseous infusion, with the proximal humerus potentially offering better puncture efficacy than the proximal tibia.