The invention discloses a method of detecting single base mutation and application. The method includes the steps that primers are designed targeted to a sample to be detected and a target segment for reference, and PCR is subjected to amplification; amplification products are subjected to balanced mixing, high-temperature denaturation is conducted, then annealing is conducted, heteroduplex DNA is obtained, then CEL I enzyme digestion is conducted, an enzyme-digested product is subjected to electrophoretic determination, and a single-base mutation sample is determined; two segments subjected to CEL I enzyme digestion are recycled, and each segment is connected with a connector provided with an A, or T, or G or C sticky tail end; one of upstream primers and downstream primers for amplification of the target segment and one of forward and reverse primers designed based on the connector sequence form two pairs of primers, each connection product serves as a template, and PCR amplification is conducted; electrophoretic detection is conducted. By means of the method for detecting single base mutation, known single base mutation can be detected, unknown single base mutation can be further screened, the position of mutation can be determined, the type of mutation can be further determined, sensitivity is high, repeatability is good, and the economic property is achieved.