Poly(l-lactic acid) (PLLA) has been extensively utilized as a biomaterial for various biomedical applications. The first and one of the most critical steps upon contact with biological fluids is the adsorption of proteins on the material's surface. Understanding the behavior of protein adsorption is vital for guiding the synthesis and preparation of PLLA for biomedical purposes. In this study, total internal reflection fluorescence microscopy was employed to investigate the adsorption of human serum albumin (HSA) on PLLA films with different molar masses. We found that molar mass affects HSA adsorption in such a way that it affects only the adsorption rate constants, but not the desorption rate constants. Additionally, we observed that HSA adsorption is spatially heterogeneous and exhibits many strong binding sites regardless of the molar mass of the PLLA films. We found that the free volume of PLLA plays a crucial role in determining its water uptake capacity and surface hydration, consequently impacting the adsorption of HSA.