This work reports a facile microfluidic device for Au-nanoparticle enhanced fluorescence detection of tiny amount of nucleotides within droplets in a high-throughput way. Droplets containing single strand DNA probe and relevant complementary strands DNA(cDNA) are generated in flow-focusing manner and the hybridization between them is realized in droplets flowing along a long serpentine channel. In order to find the optimal experimental condition, finite element method simulation is used to predict the interface evolution between the two phase liquids. Based on the fluorescence emited by intercalator reacted with the generated double-strand DNA(dsDNA), the target cDNA with a concentration of 1nM can be detected in droplets. And when we adopt Au nanoparticles to immobilize DNA probe which can amplify the fluorescence intensity, 10pM completary DNA could be detected. Due to the advantages in high-throughput and compartmentalization of this droplet platform, the detection procedure can be finished in 3 h. Our method shows good potential application in facile, sensitive, low cost and fast DNA detection for applications in personal health care.