Pancreatic enzyme replacement therapy (PERT) treats pancreatic insufficiency, causing a lack of digestive enzymes due to pancreatic damage or dysfunction. However, traditional PERT faces challenges such as poor stability and limited efficiency of the enzymes. Lipase from Porcine pancreas (PpL) is commonly applied for PERT, however only classic formulas such as tablets or capsules produced from physical mixture of the enzyme and excipients had been developed. Electrospinning produces nanofibers that are promising as drug carriers, due to their high surface area-to-volume ratio and tunable physicochemical properties. In this study we investigated the direct nanoformulation of PpL applying polymer nanofibers. The effect of water-soluble and water-resist polymers (PVA: polyvinyl alcohol, PVP: polyvinylpyrrolidone and PLA: polylactic acid) on the physicochemical properties of precursor for electrospinning and on the nanofibers produced from them was systematically investigated. The immobilization of PpL using electrospun nanofibers by entrapment and adsorption methods was developed and compared the enzymatic activities of the nanofibrous formulas. It was found that immobilization using nanofibers in all cases caused a large increase in the enzymatic activity of PpL. Our research demonstrates that nanoformulation of PpL could be efficient by electrospinning technique and the polymer has dominant effect on the enzymatic and structural properties of formulas since PpL entrapped in PLA showed the highest enzymatic activity. Nanofibrous enzyme loaded formulations were comprehensively studied by rheology, scanning electron (SEM) and Raman microscopy, water content determination, positron annihilation lifetime spectroscopy (PALS) and enzyme activity assay of ester hydrolysis.