The enzyme pepsin (E.C. 3.4.23.1, strength ≥400 μ/mg) was immobilized in poly(vinyl alcohol) (PVA) nanofibers for the first time by the electrospinning technique. The morphology of the nanofibers analyzed by SEM showed a diameter in the nanometric range of 70-165 nm. The presence of pepsin in the nanofibrous matrix was proved by infrared spectroscopy (FTIR). The immobilization process was carried out through crosslinking by exposure to glutaraldehyde (GA) vapor. The thermal analysis by differential scanning calorimetry (DSC) showed the effect of crosslinking time and enzyme concentration on the glass transition temperature (T g ) and heat of fusion (H f ) of the nanofibrous matrix. The optimum catalytic performance of the immobilized pepsin was reached at pH 3, 1 h of crosslinking and 25% of enzyme concentration. The enzymatic activity of immobilized pepsin reached 96% of the crude enzyme. The immobilization process allowed 4 cycles of reuse and up to 10 days of storage for the crosslinked samples. PVA nanofibers was successfully used as vehicle for the encapsulation and immobilization of the pepsin enzyme.