A novel method for high-performance and high-resolution separation and detection of single-stranded DNA based on low-cost plastic microchips using electrophoresis apparatus equipped with dual wavelength confocal fluorescence detection have been developed. High-quality microchannels are fabricated on a thin poly (methyl methacrylate) (PMMA) plastic sheet (0.8 mm), ensuring superior limits of detection and good dissipation of Joule heat. A reproducible allelic profiling assay for the analysis of short tandem repeat (STR) was developed using replaceable denaturing linear polyacrylamide as the sieving matrix and cellulose derivates as adsorptive modifiers to suppress nonspecific adsorption of DNA samples to the PMMA surfaces. Under optimal conditions, all fragments of the STR allelic ladders of the D13S317 and CSF1PO loci as well as PCR-amplified samples from individuals can be separated unambiguously within 180 s. These results demonstrates the potential of plastic microchips for low-cost genetic analysis.