A star-shaped fluorescent DNA probe (S-probe) is described that can recognize target DNA and discriminate it from interfering DNA via strand displacement including branch migration and toehold exchange. The artificially designed S-probe does not harm the strand displacement while it allows the fluorescently labelled strand and the quencher-labelled strand to be shared among different S-probes targeting different genetic variations. Generally, multiplexed detection of different MT/WT pairs requires different fluorophore-labelled and quencher-labelled strands. The two labelled oligonucleotides of S-probe have sequences decoupled from the target/interfering DNA sequence, so the same fluorescent and quencher strands can be used for different S-probes that target different sequences. The sensitivity, specificity, and general applicability of the method toward BRCA 41293497 mutation, KRAS G13D mutation and two types of EGFR mutations (T790 M and L858R) were experimentally demonstrated. The limit of quantification of the MT concentration is 2 nM, and the detection limit of the low abundance of the target sequence is 5% (40 nM of MT strand in the background of 760 nM of WT strand). The fluorometric assay with excitation/emission wavelengths of 485/582 nm was successfully applied to clinical samples spiked with mutant-type and wild-type DNA. The unique structure of the S-probe provides a useful tool for the regulation of the strand displacement reaction. Conceivably, the star-shaped DNA probe can be widely adopted to multiplexed detection of genetic variations and provide novel insights into the regulation of strand displacement processes as utilized in DNA based nanomachines.