Cronobacter spp. are important opportunistic foodborne pathogens in powdered infant formula that cause many serious diseases in neonates and infants. In this study, a novel assay based on dual signal amplification strategy was developed by coupling asymmetric tailing PCR (AT-PCR) with rolling circle amplification (RCA) for the detection of Cronobacter spp. in milk. The tailing single-stranded DNA was generated through AT-PCR and used to initiate RCA, generating tandem repetitive G-quadruplex sequences. In the presence of the fluorescence dye thioflavin T that could intercalate into the G-quadruplex structures, the fluorescence signal was detected with a microplate reader. The AT-PCR coupled with RCA assay was specific for Cronobacter spp. detection because of the highly specific primers chosen for the AT-PCR. The limits of detection were 4.3 × 101 cfu/mL in pure culture and 4.5 × 102 cfu/mL in spiked milk, respectively. The fixed sequences designed in the hairpin DNA allowed this AT-PCR coupled with RCA assay to serve as a universal platform for the detection of other pathogens by modifying the specificity of the PCR primers.