Here, we report two-dimensional, single-crystalline holey graphyne (HGY) synthesized in an interfacial two-solvent system through a Castro-Stephens-type coupling reaction from 1,3,5-tribromo-2,4,6-triethynylbenzene. As a new type of 2D carbon allotrope, HGY is alternately linked between benzene rings and C≡C bonds and is composed of a pattern of six-vertex and highly strained, eight-vertex rings and an equal percentage of sp2 and sp hybridized carbon atoms. By combining experimental and theoretical studies, we systematically investigated the stability of HGY and its vibrational and optical properties. Density functional theory computations predicted that HGY is a p-type semiconductor that embraces a direct bandgap (∼1.1 eV) with a high carrier mobility. Transmission electron microscopic studies revealed that the synthesized HGY sheets are highly crystalline with AB stacking. Its semiconducting character, nonlinear sp bonding, and special π-conjugated structure endow HGY with promising applications in optoelectronic, energy harvesting, gas separation, catalysis, water remediation, sensor, and energy-related fields.