The classic Langmuir-Blodgett (LB) film technique is an advanced and powerful tool to prepare the highly ordered ultrathin films at the molecular and atomic levels, whereas it cannot totally reveal the aggregation mechanism and conformation change of the Langmuir monolayers. In this work, the separate-gradient and continuous-gradient LB film methods are proposed to resolve these problems. Two kinds of gradient LB monolayer films of an asymmetric block copolymer polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA) were successfully prepared, and their structures were compared with those of the conventional single LB films by atomic force microscopy (AFM). At the air/water interface, the initial non-typical aggregates/micelles of PS-b-PMMA spontaneously form, and each copolymer molecule forms several connected intramolecular PS-rich domains (small cores) tailed/surrounded with a PMMA block/shell. Upon monolayer compression, the small PS cores coalesce or connect into large cores or rods/networks through compression-driven morphological evolution. Combining with the continuous-gradient LB films, the time-consuming “ex-situ” AFM can be regarded as a “quasi-in-situ” approach to study the interfacial structures/aggregation behavior. The above two gradient LB film methods are great contributions to the development and complementary of the classic LB film technique.