To test a new duplex nanoparticle-assisted polymerase chain reaction (PCR) on human papillomavirus (HPV) E6 oncoprotein detection of HPV16/18 positive samples from vaginal and urine self-sampling specimen. Six specimens from cervical biopsy ranging from CIN1 to CIN3, which have been testified by conventional PCR, were used for nanoPCR HPV E6 oncoprotein specificity test. Seven serially diluted templates by ten-fold (3.78 x 10(6)-3.78 x 10(0) copies/mu L) chosen from testified CIN3 biopsy were selected for the sensitivity test. Twenty self-sampling vaginal and urine samples each from 20 women in our hospital were tested by nanoPCR and conventional PCR. NanoPCR showed high specificity when detecting HPV16/18 infected CIN2 and CIN3 samples from cervical biopsy and ten-fold higher sensitivity than conventional PCR when detecting HPV16/18 infected CIN3 samples from cervical biopsy. DNA gel electrophoresis showed brighter bands on positive HPV E6 oncoprotein specimen from self-collected vaginal and urine samples compared to conventional PCR and higher clinical detection rate than those by conventional PCR on vaginal and urine self-sampling specimens. Duplex nanoPCR can be used as a better method for HPV E6 oncoprotein detection in the high-risk HPV infection group for its novelty of high specificity in low concentration target detection, as was showed in the specimen comes from vaginal and urine self-sampling, thus promoting self-sampling for better cervical cancer screening.