Visualization of single-hole nozzles into quiescent ambient has been used extensively to characterize spray mixing and combustion. However in-cylinder flow usually has some impact on the spray evolution, which may be meaningful. In the present work, the visualization of direct diesel injection spray under both non-reacting and reacting operating conditions were conducted in an optically accessible two-stroke engine equipped with a single-hole injector. Two different techniques, Schlieren imaging and UV-Light Absorption, were applied here to quantify vapor penetration for non-reacting spray. Meanwhile, Mie-scattering was used to measure the liquid length. As for reacting spray, Schlieren and OH* chemiluminescence were applied simultaneously to obtain the spray tip penetration and flame lift-off length under the same TDC density and temperature. Additionally, PIV was used to characterize in-cylinder flow motion. Results were compared with ECN database which were obtained under quiescent ambient conditions in a high pressure high temperature vessel. Because of the air flow and piston movement, in-cylinder conditions in the two-stroke engine during the spray injection are unsteady, which has a significant impact on the spray development and interference on the spray visualization. It was found from the comparison with ECN data that the air flow caused by the piston movement can slow down vapor penetration. Moreover, both ignition delay and lift-off length under unsteady conditions show less sensitivity with ambient temperature sweep than that of quasi-steady conditions.