Single base mutations are closely related to cancer diagnosis and treatment. The fluorescent probe method is one of the important methods to detect single-base mutations. We constructed a universal probe detection system based on endonuclease IV and the DNA strand displacement reaction. The system uses two toehold strand displacement reactions to relay the mutation information to the universal strand. There is no need to design the probe one-by-one for each mutation point during multi-site detection. It has the advantages of simple operation, rapid detection, and low cost. We used this method to detect common clinical mutation sites (PTEN R130Q/EGFR L858R/PTEN rs1473918395), and the detection limit can reach 0.1%-1%. The detection system can provide a new rapid and economical method for clinical single-base mutation detection, and has broad application prospects in diagnosis and prognostic evaluation.