This study achieved single molecule detection, for the first time, on the prefabricated substrate for SERS (surface enhanced Raman spectroscopy) with non-resonant molecules. The substrate with directionally arrayed gold nanoparticle dimers enables huge Raman enhancement and is expected for single molecule identification and structural analysis of a wide range of bio/chemical molecules. Due to remarkable capabilities of rapid and highly-sensitive structural analysis and identification of bio/chemical molecules, SERS is expected as a powerful tool for single molecule analysis. Conventionally molecularly bridged metal nanoparticle aggregates has been used for single molecule SERS so far. In these demonstrations, the aggregates are generated mixing nanoparticle colloid and target analyte solutions with hour-level long incubation time. Rhodamine 6G or crystal violet, which offers a resonant Raman effect, has been used. Therefore, for example, this technique cannot be applied to in-situ real-time analysis of various bio/chemical molecules such as hazardous substances, cell products and nucleobases in microfluidic devices. Although prefabricated SERS structures such as particle aggregates on a substrate are appropriate for the above application, no demonstration of single molecule SERS has been reported. It is because the reported SERS substrate has an insufficient Raman enhancementfactor for single molecule SERS. In this study, for the first time, we demonstrate single molecule SERS of non-resonant molecules using the prefabricated substrate. In order to improve Raman enhancement factor of SERS substrates, this study uses the nano-engineered SERS substrate on which particle dimers are directionally arrayed to match all dimers axes to a polarization direction of incident light. This arrangement is expected to enable single-molecule SERS analysis, because of the large Raman enhancement achieved by coupling with the polarization direction. Arraying the dimers directionally, the huge enhancement can be achieved at all dimers. The substrate was fabricated using nanotrench-guided self-assembly. At the beginning of this fabrication, a colloidal particle solution was injected between a cover glass and a template substrate of Si with nanotrench template. On drying the aqueous particle dispersion between the substrates, the water surface line moved backward and the particles became concentrated near the edge of the meniscus. The drag force pressed the particles onto the template substrate. When the meniscus passed over the templates, particles were trapped on the template. Gold nanoparticles with mean diameters of 100 nm were used in this study. 4,4'-Bipyridine molecule, which is non-Raman resonance molecule and a pesticide material, was used for demonstrating single molecule detection. We performed around 50 Raman measurements for each measurement time (1 s or 0.05 s) and molecule concentration (10−5 or 10−11 M). 10−11 M corresponds to one molecule per volume of a cube with a side 5.5 µm. The statistical analysis was performed. All spectra were discriminated based on with or without a peak at around 1609 cm−1, which is one of the Raman shifts derived from the target molecule. At 10−5 M concentration, all spectra exhibited clear peaks. The distribution of Raman intensities were fitted by one Gaussian curve. At 10−11 M concentration, the experimental data at 1 s and 0.05 s were fitted by three and two Gaussian curves, respectively. These are consistent with a Poisson distribution. The change in the statistical distribution from Gaussian (10−5 M) to Poisson (10−11 M) with decreasing the concentration reflects the probability of detecting 0 (background noise), 1, 2 molecule(s) at 1 s. Two Gaussian curves indicate 0 and 1 molecule at 0.05 s. Average relative intensities of 0, 1 and 2 molecule(s) were 0.89, 1.06, and 1.28 at 1 s, respectively. The net relative intensities of 2 molecules are calculated to 1.28−0.89=0.39, which is 1.8 times as much as 1 molecule, 1.28−1.06=0.22. From these experimental data and statistical analysis, we confirmed that the developed substrates achieved single molecule SERS detection. Finally, nucleobases identification with single molecule sensitivity was demonstrated using 10−11 M solutions of adenine and cytosine. The obtained peaks were enough clear to identify the nucleobases. Therefore, a wide range of bio/chemical molecules is expected to be structurally analyzed and identified by single molecule SERS on the prefabricated substrate.