For technological purposes there is an urge towards the fundamental knowledge of biomolecules-clay interaction at a single molecular level. To this aim, in this work we investigated in detail the specific interaction between the basic amino acid l-alanine and the (001) clinochlore surface. The adsorption process of alanine was investigated by cross-correlated atomic force microscopy (AFM) and quantum mechanics simulations (QM) at the DFT/B3LYP level of theory. As similarly deduced for glycine, clinochlore surface was able to condense, organize, and agglomerate alanine molecules onto specific regions, the brucite-like ones. The amino acid was stably adsorbed onto the brucite-like surface of clinochlore, organized in dot-like structures, agglomerates and filament-like structures up to about 150 nm long. Single molecules of alanine were discriminated by AFM, with sizes in agreement with QM calculations (mean height of about 5 ± 0.5 Å and a mean width of about 11 ± 1 nm). Simulations provided also the lowest energy conformation of the molecule on the surface, the intermolecular bonding scheme and the amino acid/surface interaction energy, which could be as high as −250 kJ/mol. To further extend the knowledge of the specificity of the brucite-like sheet to interact with biomolecules, dedicated quantum mechanical investigations were also conducted to quantify the Brønsted−Lowry basic strength related to Al3+/Mg2+ substitutions in the brucite-like sheet, typically occurring in this kind of clay mineral, simulating the interaction with probe molecules with increasing acidic strength (H2O, H2CO3, HNO3 and HCl). The reported findings could be useful not only for biotechnological and environmental purposes, but also for prebiotic chemistry research, because clinochlore is one of the 420 mineral species likely to have been present in Earth's near-surface environment at the time of life's origins (Hazen, 2013).