Main observation and conclusion Endonuclease IV (Endo IV) has the side activity to cleave the apurinic/apyrimidinic site in the single-stranded DNA probe (AP-ssDNA), which adversely affects its performance in single-nucleotide variation (SNV) detection. In this work, we developed a simple strategy to inhibit Endo IV's side activity by introducing an assistant strand into the detection system. The assistant strand effectively inhibited the side-activity of Endo IV by confining the freedom of AP-ssDNA through rigid double-stranded DNA formation, and the inhibition efficiency could reach 98%. About 20 times enhancement of the discrimination factor (DF) was obtained compared to the detection system without the assistant strand. After optimization, a mean DF value was calculated to be 738 for different mutation types, and the sample with 0.005% allele frequency was discriminated from the wild-type target. Another advantage of using an assistant strand was that the SNV detection could be executed at physiological temperature without precise temperature optimization as in other Endo IV-based SNV detection system. Besides, EGFR T790M mutant in the synthesized and clinical samples was detected by our strategy. The results showed satisfactory sensitivity and accuracy by comparison with Sanger sequencing. Thus, this strategy has the potential to be applied in the field of precision medicine.