A highly sensitive and selective multi-walled carbon nanotube (MWCNT)-based multicolor fluorescent nanobeacon is developed for multiplexed analysis of DNA in homogeneous solution. In this work, three different dye-labeled DNA hairpins were adsorbed on MWCNTs surface via π-stacking, which brings the dyes and MWCNTs into close proximity and leads to the quenching of fluorescence of the dyes. When target DNAs were added to the solution, the target DNAs specifically hybridize with the probes to form stable DNA duplexes, which weakens the interactions between the probes and MWCNTs, and results in the fluorescence recovery of the dyes. By using three 15-mer DNA fragments as proof-of-principle analytes, the proposed method showed good analytical performance. The limits of detection obtained were in the range of 35–42pM. Moreover, this method also exhibits an excellent ability to discriminate between single nucleotide polymorphisms.