The combination of two in situ techniques for the study of immobilization of biomolecules is seldom performed. Therefore, the combination of in situ ellipsometry with atomic force microscopy (AFM) in a liquid was used to follow the adsorption process of enolase, an enzyme of the glycolytic pathway. The adsorbed amount and topographic information of the adsorbed layers were obtained in the liquid environment. AFM images showed that surfaces with comparable adsorbed amounts of protein presented immobilized entities arranged in strings or in spherical clumps, evidencing the importance of combining in situ ellipsometry and AFM in a liquid.