Single-molecule tracking (SMT) was used to probe the alignment of cylindrical microdomains in polystyrene–poly(ethylene oxide) diblock copolymer (PS-b-PEO) films induced by directional solvent penetration. PS-b-PEO films were supported between two glass plates. Aligned PEO domains were obtained upon horizontal penetration of 1,4-dioxane vapor through the films, while no observable alignment occurred when benzene or toluene was used. Domain alignment was assessed by SMT, using a dye that partitions into PEO. More than 70% of the dye molecules exhibited one-dimensional (1D) diffusion along the dioxane penetration direction. Quantitative assessment of 1D trajectory alignment yielded an order parameter of ca. 0.9. A high degree of domain order was observed across millimeter distances throughout the 4 μm thick films. PEO domain size (ca. 11 nm radius) was determined from the probe positioning error. These results demonstrate the utility of SMT methods for characterization of nanoscale domains in cylinder-forming block copolymer films.