In this work, a novel amperometric biosensor for the determination of adenosine triphosphate (ATP) was developed. The biosensor was based on two enzymes (glucose oxidase and hexokinase) that were entrapped in the electrospun nanofibers. The biosensor was prepared by direct electrospinning of the enzymes/polymer mixture on the surface of platinum disc electrodes with subsequent UV cross-linking of the obtained nanofibers. Photocrosslinkable polymer (polyvinyl alcohol containing styrylpyridinium groups, PVA-SbQ) was chosen as matrix, while gold nanoparticles (GNPs) were added to the enzymes/polymer mixture to improve analytical characteristics of the biosensor. For the first time, incorporation of several enzymes in the nanofibers during electrospinning was demonstrated. Composition of the mixture for electrospinning was optimized. The obtained biosensors enabled successful detection of ATP by chronoamperometry, linear range was 25-200 μM. The analytical characteristics of the biosensors were compared with the biosensors prepared by manual drop-casting of the enzymes/polymer/GNP mixture. The biosensor can be also used to detect glucose in samples with known or very low (<10 μM) ATP concentration; linear range of the glucose determination was 0.01-3 mM. The described approach for one-step electrospinning of several enzymes is fast and simple and can be easily modified for immobilization of other multi-enzyme systems, which opens possibilities to enlarge the range of the targets that can be determined by the biosensors produced using electrospinning.