The present study was aimed at the preparation and characterization of tailor made hybrid materials, whose peculiar hosting capability could be exploited in biotechnological applications. With this purpose, the modification of K10 montmorillonite by intercalation of Tween 20 surfactant, was accomplished. The influence of two internal parameters, namely pH and surfactant/clay ratio, on the surfactant uptake ability by clay was investigated. The adsorption mechanism was elucidated on the basis of complementary kinetic and equilibrium studies and, then, corroborated by the useful information provided by the FT-IR, TGA and XRD characterization. The gathered results allow to draw the conclusion that the whole surfactant uptake is the result of two contributions: a site-limiting component, governed by negative cooperative interactions, which takes into account for the Tween 20 adsorption onto the pristine clay, and a non-specific linear partitioning component, related to the adsorption of the surfactant onto the in situ prepared organo-clay. Moreover, at strongly acidic pH, a mechanism consisting of two-steps pathways involving two non-energetically equivalent binding sites of the clay surfaces, was proposed, while, on increasing the pH, the clay interlayer becomes the sole available site for the surfactant uptake. In the light of the interesting results obtained, among the plethora of potential biotechnological applications, the present paper suggests the exploitation of the prepared organo-clays to improve the performance of either hydrophilic or hydrophobic drug carriers systems