The understanding of the adsorption process of biomolecules is very important for biological and engineering applications. Enolase is an enzyme of glycolytic pathway that catalyses a reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate. In this work the adsorption behavior of enolase (2-phospho-D-glycerayte hydrolyase) onto hydrophilic silicon wafers and amino-terminated surfaces (APS) and onto hydrophobic polymer polystyrene (PS) was studied by means of null-ellipsometry. The adsorption kinetics of enolase onto these substrates presented three distinct regions: W a diffusion-controlled one; (ii) an adsorption plateau; (iii) continuous, irreversible, and asymptotic increase of the adsorbed amount with time. Atomic force microscopy (AFM) showed that well-packed entities formed an enolase biofilm, which might correspond to the monolayer formation. With increase of the adsorption time, aggregates appeared on the surface, suggesting multilayer formation. The early stages might be predicted by the random sequential adsorption model (RSA), while the cooperative sequential adsorption (CSA) model seems to describe regions ii and iii. No significant influence of ionic strength was observed on the adsorption behavior of enolase onto the present substrates. The adsorption isotherms show that enolase has no preferential adhesion onto hydrophilic or hydrophobic substrates. Contact angle measurements showed that PS surfaces became hydrophilic and silicon surfaces turned hydrophobic after the formation of the enolase biofilm. The study of the influence of pH on the enolase adsorption on silicon and APS surfaces showed that the higher adsorbed amount occurs when pH is close to enolase pI. Far from pI the enzyme solubility decreases and some repulsive forces come out, leading to a decrease in the adsorbed amount.