Transparent sol-gel thin films immobilizedwith human serumalbumin (HSA) were made via the acid catalyzed sol-gel reaction of tetraethylorthosilicate in presence of the human serum albumin. Different surfactants include; cationic cetyl trimethyl ammoniumbromide (CTAB), anionic sodiumdodecyl sulfate (SDS) and nonionic Triton X-100 (TX-100) were tested for the improvement of the host material mesostructure, increasing its porosity and well accommodation of the HSA protein within the silica matrix. The thin films show similar behavior in presence of surfactants as their free counterparts in aqueous solution with a significant shift in the wavelength of absorption. The activity behavior of the immobilized human serum albumin retained its activity by immobilization.Different parameters include concentration of protein and surfactant, type of surfactant, life time and number of measurements were investigated. The HSA thin film sensor showed stability, repeatability, reproducibility and long life time behavior. Maximumstability of HSA thin films were achieved by drying at 37 oC.