Chitosan-blended cellulose monoacetate nanofibers were prepared through electrospinning process. Neat nanofibers and their sodium hydroxide-treated analogs were used as support surfaces for protease immobilization via physical adsorption method. Morphologies of the nanofibers were observed with a scanning electron microscopy. Chemical analyses were conducted with Fourier transform infrared spectroscopy, and thermal analyses were carried out with differential scanning calorimeter and thermogravimetric analyzer. Immobilized enzyme activities were measured by using casein substrate. In order to test the stability of immobilized enzymes, the tests were repeated until the immobilized enzyme activity was leveled off. The results reveal that well uniform cellulose monoacetate/chitosan nanofibers were obtained, and nanofiber structures are transformed from rounded to more flattened morphology after enzyme activation test. Glutaraldehyde activation has positive effect on sodium hydroxide-treated samples, and the highest immobilization yield as about 83% was observed for glutaraldehyde-treated cellulose monoacetate/chitosan samples. Sodium hydroxide treatment before glutaraldehyde activation shows the best results for protease immobilization on cellulose monoacetate and cellulose monoacetate/chitosan nanofibers. Operational stability increases after sodium hydroxide treatment and glutaraldehyde activation. Glutaraldehyde activation effectively increased the cycle number after sodium hydroxide treatment and about 20% of enzyme activity was still retained after seven cycles at cellulose monoacetate/chitosan samples. This percentage is higher at pure cellulose monoacetate nanofibers than cellulose monoacetate/chitosan nanofibers and measured around 33.5%.