Polymerase chain reaction (PCR) is a very powerful tool for clinical gene detection. Multiplex PCR especially improves the throughput of this technology. However, it is often necessary to employ techniques such as electrophoresis, mass spectrometry, or sequencing after multiplex PCR amplification for product identification, which requires additional equipment and has high risks of contamination. In this work, we developed a high-throughput two-dimensional (2D) PCR technology that can identify multiple target genes simultaneously in just one closed tube and within a relatively short time by using both fluorescence and the melting temperature (Tm). As an example, a method detecting 9 human papillomavirus (HPV) subtypes and reference genes in a single tube was successfully established using 2D PCR. If designed properly, 2D PCR is believed to have the capability to identify more than 30 genes in one closed tube at a time. This method is particularly suitable for distinguishing microorganisms, single-nucleotide polymorphisms, and the methylation of genes and will be of great help to clinical work.