The invention relates to the chromosome deletion detection field, and especially relates to a multiple real-time fluorescent PCR detection kit for Y chromosome microdeletion. The kit detects gene deletion at sY84 and sY86 loci of a Y chromosome AZFa subregion, sY127 and sY134 loci of a Y chromosome AZFb subregion, and sY254 and sY255 loci of a Y chromosome AZFc subregion by real-time fluorescent PCR. The kit realizes a stable and high-efficient multiple PCR reaction system by using homologous tailed primers and universal primers, adopts nucleic acid probes labeled by different fluorescent genes to indicate the presence of target fragments, and thus realizes the fastest simultaneous detection of sequences on 6 AZF deletion hotspots and 2 internally controlled genes by two tubes. The kit of the invention can be used for detection of male Y chromosome microdeletion genes, and has the advantages of high speed, accuracy, high throughput, low cost, no pollution, and the like.