The aim of this study was to synthesize dual-functionalized poly(vinyl alcohol) (PVA)/poly(acrylic acid) (PAA) electrospun nanofibers with enzyme and copper ion (Cu(II)) for enhancing anti-biofouling activities. The PVA/PAA nanofibers were successfully synthesized by co-electrospinning (voltage=17kV; tip-to-collector distance=15cm) and cross-linked by heat treatment. The PVA/PAA nanofibers were functionalized through adsorbing Cu(II) onto the nanofibers to prepare the PVA/PAA-Cu(II) nanofibers. Three proteases (proteinase K, trypsin, and -chymotrypsin) and a quorum quenching enzyme (acylase I) were tested for biofilm reduction that -chymotrypsin effectively inhibited the biofilm formation and removed biofilms of Pseudomonas aeruginosa and Staphylococcus aureus. The PVA/PAA nanofibers were dual-functionalized with -chymotrypsin and Cu(II) to obtain PVA/PAA-Cu(II)- nanofibers. Degradation tests for extracellular polymeric substances (EPS) extracted from P. aeruginosa indicated that the PVA/PAA-Cu(II)- nanofibers could degrade the EPS proteins up to 0.26mgmL(-1) for 300min, which was higher than that of free -chymotrypsin. For anti-biofouling tests, the log number of planktonic and sessile cells of P. aeruginosa was the lowest in the PVA/PAA-Cu(II)- nanofibers. The anti-biofouling activities of the PVA/PAA-Cu(II)- nanofibers could be attributed to the effects of both Cu(II) (killing planktonic and sessile cells) and -chymotrypsin (degrading the EPS protein in biofilm).