Here we report a rapid, low cost, and disposable dipstick-type DNA biosensor that enables multiplex detection in a single assay. The fluorescent probes labeled with different fluorophores were introduced into the lateral flow nucleic acid testing system. In combination with multiple immobilized probes arranged in an array formant on the membrane, a dual-color fluorescent lateral flow DNA biosensor was developed using a portable fluorescence reader. Up to 13 human papillomavirus types could be detected simultaneously by a single-step operation in less than 30 min after linear-after-the-exponential (LATE)-PCR. The sensitivity was determined to be 10-10(2) copies plasmid DNA/μL. The specificity study showed no cross-reactivity among the 31 different common HPV types. In the clinical validation, 95.3% overall agreement showed very good potential for this method in the clinical application when compared to a commercial kit.