Self-assembled monolayers are the basis for molecular nanodevices, flexible surface functionalization and dip-pen nanolithography. Yet self-assembled monolayers are typically created by a rather inefficient process involving thermally driven tethering reactions of precursor molecules to a metal surface, followed by a slow and defect-prone molecular reorganization. Here we demonstrate a non-thermal control over the self-assembly of phenylacetylene on gold that produces previously unachievable well-ordered three-dimensional monolayers, where the molecules are attached directly through the alkyne group. While thermal excitation can only desorb the parent molecule due to prohibitively high activation barriers for attachment, localized injection of hot electrons or holes not only overcomes this problem, but also enables an unprecedented control over subsequent ordering of attached molecules on the surface, including a nanoscale control over the size and shape of the self-assembly, defect structures and the reversible process between flatlying and upright molecular configuration from single molecular level to mesoscopic scale. This work thus demonstrates the feasibility of non-thermal reaction pathways that may lead to unique and controllable self-assembly in supported molecular layers.