Si lattice-based linewidth, as the most used calibrator in the semiconductor industry, failed to meet the nanoscale demands of critical dimensions (CD). In 2018, the Si{220} lattice spacing was recommended as a secondary realization of the meter definition, however, Si lattices as nanoscale rulers can only be used in ultra-high vacuum (UHV) environments. For the first time, we designed two-dimensional (2D) supramolecular self-assembly monolayers (SAMs) as a new generation of nanoscale rulers using scanning tunneling microscopy (STM) technology in atmospheric environment. Three SAMs nanoscale rulers were selected among seven types of SAMs based on their self-assembly behaviors and thermodynamic stability. One of these SAMs nanoscale rulers was used to calibrate the STM images through lattice perspective transformation method, and directly determine the lattice length along the nanoscale rulers’ lattice direction without calibration, proving the feasibility of SAMs nanoscale rulers in nanometrology. The construction of SAMs nanoscale rulers was straightforward and manageable in the atmosphere, significantly lowering the complexity of producing nano precise measurement tools. Identifying the ideal SAMs for use as nanoscale rulers holds immense potential to significantly reduce the cost of calibration in chip industries.