Enzymes can be used multiple times when they are immobilized on a support. More enzymes can be immobilized on a surface when nanofibers are used as a supporting surface because the specific surface area increases tremendously. In this regard, polycaprolactam/cellulose monoacetate (PA6/CMA) and polycaprolactone/cellulose monoacetate (PCL/CMA) blended nanofibers (NFs) were prepared via an electrospinning process. Protease enzymes were immobilized on neat PA6, PCL, PA6/CMA, and PCL/CMA nanofibers and glutaraldehyde (GA) activated analogs through the physical adsorption method. The immobilized enzyme activity was measured by using a casein substrate, and the results were compared with free enzyme activity. Among all of the samples, the highest immobilization yield of about 82% was obtained with GA-activated neat PCL NF samples. The best remaining activity of the immobilized enzymes on pure CMA NFs was found to be 59% after seven reuses. Even after nine reuses, enzyme activities are still observed on the CMA NF samples. It was expected that the addition of CMA in PCL and PA6 NFs would increase the reusability number to reach the reusability of CMA NFs, but it was not significantly enhanced. If CMA chains could be mostly collected on the sheath or close to the sheath of the NFs during the electrospinnig process, this target could be achieved. (c) 2017 Wiley Periodicals, Inc.