AIMS To explore whether the first or the second drop of blood is more suitable for the self-monitoring of blood glucose (SMBG). METHODS SMBG was employed in hospitalized patients using the first and second drop of blood. Venous blood glucose was measured meanwhile. The differences in blood glucose measurements were then compared in groups with different regions of blood glucose levels. RESULTS There were 802 groups of blood glucose in 526 patients. There was no significant difference in the blood glucose levels of the first and second drop of blood and venous blood. However, after combining then dividing measurements into six groups according to blood glucose concentration, we found statistically significant differences between the blood glucose levels obtained from the first drop, second drop, and venous blood in the groups containing blood glucose values <9.9 or 20-30 mmol/L. In contrast, there were no significant differences in the 10-14.9 or 15-19.9 mmol/L groups. CONCLUSIONS In the clinical setting, both the first or second drop of blood can be used for performing SMBG to assess real-time venous glucose. By categorizing blood glucose into different levels more accurately, we observed that there was no significant difference between the first or second drop of blood and the venous blood glucose value when blood glucose levels were maintained between 10 and 20 mmol/L. When blood glucose levels were below 10 mmol/L, the value obtained from first drop of blood was close to that from venous blood, whereas when the blood glucose level is >20 mmol/L, the blood glucose value from the second drop of blood was more accurate.