 We developed a novel method called DNA fragment mass fingerprinting, DFMF, to rapidly identify and distinguish different DNA sequences. This technique utilizes aqueous micro droplets to accelerate deoxyribonuclease I, DNAs I, mediated DNA fragmentation, which can be achieved in under one millisecond. Furthermore, this method employs a cosine correlation measure to compare the master charge ratio, MZ, of each fragment's peak relative intensity and base pair sequence similarity. This allows for the rapid identification of single nucleotide mutations, such as those found in the sickle selenemia mutation, with a similarity score of 90%. Additionally, this method can also differentiate two adjacent bases in the sequence with a similarity score of 62%, which is significantly lower than the 98% similarity score required for gel electrophoresis or direct mass detection by mass spectrometry. In comparison to traditional genotyping methods, such as quantitative real-time polymerase chain reaction, DFMF can complete the identification process within minutes without requiring additional reagents or complex experimental procedures. Finally, this method has been successfully used to differenti.