The synthesis of decanoate β-cyclodextrin esters (β-CDd) and hexanoate β-cyclodextrin esters (β-CDh) was biocatalyzed by thermolysin from native β-cyclodextrin (β-CD) and vinyl hexanoate or vinyl decanoate used as acyl donors. The products were chemically characterized by infrared, NMR, and mass spectrometry. Both β-CDd and β-CDh esters were identified as a mixture of β-CD preferentially substituted on the C2 position by the corresponding acyl chain. The degree of substitution varied from 2 to 7 for β-CDd and from 4 to 8 for β-CDh. The ability of β-CD esters to self-organize into nanoparticles was tested using a nanoprecipitation technique in various solvents. The mean size diameter and polydispersity measured by quasi-elastic light scattering were dramatically affected by the nature of solvent (acetone, ethanol, or tetrahydrofuran) used in the nanoprecipitation technique. When directly observed using cryo-transmission electron microscopy, β-CDh appeared as uniformly dense nanospheres, whereas β-CDd exhibited a multilamellar onion-like organization. A structural model was rationalized for the β-CDd nanoparticles.