Cationic polyelectrolyte doped hollow nanofibers prepared via facial coaxial electrospinning technology have been used for positional assembly of two enzymes, glucose oxidase (GOD) and Candida antactica lipase B (CALB), at two different positions, namely, in their lumen and on their surface. Therefore, the result is four combinations, including lumen (GOD+CALB), surface (GOD+CALB), surface (GOD)-lumen (CALB), and lumen (GOD)-surface (CALB). Surface attachment of enzymes was achieved by layer-by-layer (LbL) technology, which is based on the ion-exchange interactions between oppositely charged enzymes and polyelectrolyte that was doped in hollow nanofibers; whereas placing enzymes inside the lumen of hollow nanofibers was realized by in situ encapsulation during coelectrospinning. The hollow nanofibers-based biencyine systems were used for a cascade reaction in an oil-aqueous biphasic system, in which glucose was oxidized by GOD to generate H2O2, which was used as substrate and oxidant for CALB-catalyzed epoxidation of oleic acid in the second step. The bienzyme nanofibers membrane was found to float spontaneously at the O/W interface, which is advantageous to biphasic biocatalysis. Assembly strategies of the two enzymes affect their biocatalytic efficiency significantly by influencing the utilization efficiency of H2O2 in the reaction process. The highest reaction rate was attained by lumen (GOD)-surface (CALB), corresponding to 114.45 times enhancement as compared to that of the free bienzyme system.