Acetylene black is a special type of carbon black, obtained from the thermal decomposition of acetylene. Acetylene flames enable the investigation of the soot optical properties. A reliable knowledge of the optical properties of acetylene soot is necessary for interpreting the optical measurements in flames and the development of acetylene black applications in industry. However, a wide variation in the optical properties of soot is observed in different formation conditions. The main factors affecting soot formation in flames are fuel composition, equivalence ratio, and reaction time connected with the height above a burner. In this study, we measured the absolute value of the refractive index function of acetylene soot E(m,1064) at a wavelength of 1064 nm, as well as the ratio of the refractive index functions E(m,1064) / E(m,532) at laser wavelengths of 532 nm and 1064 nm in a premixed acetylene/air flame using laser-induced incandescence. The soot structure was studied using high-resolution electron microscopy. The obtained results showed that E(m,1064) increased from 0.19 to 0.53 and that the ratio of E(m,1064) / E(m,532) increased from 1.12 to 1.55 according to the height above a burner. These changes in the optical properties are attributed to differences in soot structure, such as different distances between graphene planes inside soot crystallites, which decreased from 0.43 to 0.36 nm as the height above the burner increased from 5 to 20 mm. It was concluded that the optical properties and structure of acetylene soot differ from those formed in other hydrocarbon flames.