Microstructural characteristic lengths in solid metals refers to density of defects that determine the mechanical properties of materials including grain size, twin spacing and initial dislocation density. Nanoporous gold (np-Au), which has large free volume and irregular ligament size and porous structure, shows different deformation behavior from solid metals, nanopillars and nanowires with regular cylindrical shape. Thus, effect of microstructural characteristic length in np-Au would be act differently and it should be reconsidered. Np-Au is synthesized by dealloying process, where sacrificial Ag in precursor alloy is selectively etched leaving Au adatoms and they make interconnected open-cell structure. We develop fabrication techniques of np-Au with nanotwinned structure by controlling internal microstructure of Ag-Au precursor alloy. Microstructures of each samples are investigated using electron microscopes before and after dealloying. We investigate correlation between process conditions and microstructure in np-Au comprehensively. Also, mechanical properties of np-Au with different microstructure are measured by nanomechanical tests such as nanoindentation and in-situ tensile tests.