The aggregation behavior of amphiphilic block copolymer polystyrene-b-poly(acrylic acid) (PS-b-PAA) at the air–water interface was investigated through surface pressure measurements (isotherms, compression–expansion hysteresis and compression relaxation experiments), Brewster Angle Microscopy (BAM) imaging and Atomic Force Microscopy (AFM) imaging. It is found that PS-b-PAA (M_n = 11490 g mol⁻¹, PAA wt%62%) forms a stable Langmuir monolayer on the water surface (pH = 7) by using N,N-dimethylformamide (DMF) as the spreading solvent. The aggregation of block copolymer is induced by an initial diffusion of DMF into water from the interface. Upon compression of the film, the pseudoplateau observed in the Langmuir isotherm corresponds to a “pancake-to-brush” transition with the PAA chains gradually dissolving in the water subphase and stretching underneath the PS cores. Based on the isotherms and the BAM images, it is suggested that the polymer chain dynamics in spreading solutions with different concentrations at the time of solvent diffusion influence the interfacial behavior of block copolymers significantly. The Langmuir–Blodgett (LB) films prepared at different surface pressures from different spreading solution concentrations were scanned by AFM. A variety of morphologies such as wormlike, porous and reticulate structures, and dots were observed. The isotherms and AFM images show the spreading solution concentration and surface pressure dependence of the aggregation behavior of PS-b-PAA copolymer at the air–water interface.