Objective To establish an efficient DNA extraction method for the batch identification of genotypes in model mice. Methods We extracted total DNA from transgenic mouse tails using four Methods: alkaline simplified group, alkaline routine group, protease K cleavage group and DNA extraction kit group. The purity and concentration of DNA obtained by the four Methods were measured, the effects of gel electrophoresis were evaluated, and the time and experimental costs of the four Methods were compared. Results The protease K cleavage method produced the highest concentration of DNA, followed by the simplified alkaline boiling and routine alkaline boiling method. The reagent kit produced the highest DNA purity, followed by the simplified alkaline boiling method. The DNA templates obtained by the four method could be amplified by polymerase chain reaction and gel electrophoresis to obtain clear DNA target bands. In addition, the DNA template extracted by the simplified alkaline boiling method could be used for gene identification after storage at -20℃ for 1 month, as well as requiring the least time and lowest costs. Conclusions The simplified alkaline boiling method is currently the simplest, fastest, and most economical DNA template-extraction method for batch identification of genotypes in model mice.