Objective Four different methods were examined to identify a safer and more reliable method for tail vein punctures in C57BL/6 mice. Methods In total, 320 mice were randomly divided into four groups: a blank group, incandescent lamp baking method group, three-line method group, and combined method group. Blood samples were taken from the left or right peripheral vein of puncture mice. Puncture success rate of each group was recorded. SPSS 13.0 software was used to compare statistical difference among groups. Results Compared with the blank group, success rates of the other three methods were significantly higher ( P < 0.001). Further, the three-line method was better than the incandescent lamp baking method ( P < 0.001). The success rate of the combined method was significantly higher than the three-line and incandescent lamp baking methods ( P < 0.001). Conclusions The combined method greatly improved the success rate of tail vein punctures in C57BL/6 mice. This method is more reliable and should be more widely used in the future.