The current study reports on a cross-priming amplification (CPA) scheme that utilizes antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) for simultaneous detection of nucleic acids and prevention of carryover contamination. Amplification products were applied in a nanoparticle-based lateral flow biosensor (LFB). The method shows attractive features in that it only requires the use of a labeled primer, eliminating the use of labeled probes. Thus, it is able to remove false-positive results yielded by undesired hybridization between two labeled primers or between a probe and labeled primer. CPA amplification and AUDG cleavage are carried out in a single pot, and the use of a closed-vessel reaction eliminates unwanted results due to carryover contamination. Then, the assay devised in this report was applied to the detection of the hospital-acquired pathogen Klebsiella pneumoniae in pure cultures and artificial sputum samples. This biosensor can detect K. pneumoniae in pure cultures with a 100 fg . L-1 detection limit, and in artificial sputum samples with a 520 cfu . mL(-1) detection limit. The whole procedure, including specimen processing (20-min), CPA amplification (60-min), AUDG digestion (5-min) and result indicating (within 2-min), can be completed within 1.5 h. As a proof-of-concept technique, this method can be used for detecting a wide variety of other targets if the specific CPA primer set is available.