A method for the detection of lactate using the lactate dehydrogenase enzyme (LDH) non-covalently labelled with 8-anilino-1-naphthalene sulfonic acid (ANS) is described in this work. The enzyme was immobilized in poly(vinyl alcohol) (PVA) nanofibers using the electrospinning technique. The presence of the LDH-ANS system in the PVA fibers was confirmed by laser scanning confocal microscopy. According to scanning electron microscopy analysis, the PVA electrospun nano-fibers containing the labelled enzyme showed a free defects morphology, with a diameter distribution in the range of 98-115 nm. The ANS-labelled LDH system shows a decrease in the fluorescence intensity upon binding lactate. This fluorescence quenching was used to detect the presence of lactate in the reaction medium. The immobilized LDH-ANS system shows sensibility toward lactate for concentrations as low as 22 µg/mL.