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The fluorescent DNA probes using CdTe quantum dots (QDs) and Au nanoparticles (AuNPs) had complicated microstructures because one QD or AuNP can be conjugated to more than one single-stranded DNA (ssDNA) on its surface. In this Letter, an asymmetrical synthesis method of DNA probes was presented to get the DNA probes into the uniform microstructure when poly(N-isopropylacrylamide-co-divinylbenzene) microspheres (PNDMs) were used as templates. The sterically restricted linkages of CdTe QDs with ssDNA (CdTe-DNA1) and AuNPs with complementary ssDNA (Au-DNA2) were both present on PNDMs. After the CdTe-DNA1 and Au-DNA2 were cleaved from carriers and hybridised with each other, the fluorescent DNA probes (CdTe-dsDNA-Au) were prepared using 1 QD with 1 AuNP microstructure and the corresponding quenching efficiency of probes was 64.8%.