In this article, we describe an ultrasensitive nucleic acid biosensor (NAB) based on horseradish peroxidase (HRP)-gold nanoparticle (Au-NP) dual labels and lateral flow strip biosensor (LFSB). The results presented here expand on prior work (Mao et al., 2009a) by optimizing the preparation of HRP-Au-NP-DNA conjugates. It was found that sodium dodecyl sulfate (SDS) and the immobilization sequence of thiolated DNA and HRP on the Au-NP surface played very important roles to improve the sensitivity of the assay. After systematic optimization, the detection limit of current approach is 1000 times lower than that in prior work. Deposition of insoluble enzymatic catalytic product (red colored chromogen) on the captured Au-NPs at the test zone of LFSB offers a dramatic visual enhancement. Combining enzyme catalytic amplification with unique optical properties of Au-NPs, the NAB was capable of detecting of 0.01-pM target DNA without instrumentation. The NAB thus provides a rapid, sensitive, low-cost tool for the detection of nucleic acid samples. It shows great promise for in-field and point-of-care diagnosis of genetic diseases and for the detection of infectious agents.