Objective To explore the preparation of nanofiber membrane by human umbilical cord Wharton’s jelly and to explore its feasibility as a scaffold for cartilage tissue engineering. Methods The human umbilical cord Wharton’s jelly was decellularized and mixed with polycaprolactone(PCL) to prepare an electrospun nanofiber membrane. The cytocompatibility of the nanofiber membrane was verified by implantation with chondrocytes derived from rabbit ear in vitro. Thereafter, the chondrocytes-nanofiber membrane constructs were implanted in nude mice for 6 weeks to verify the feasibility of cartilage formation in vivo by histological examination. Results After decellularization of Wharton’s jelly, the cellular components were removed and most of the collagen was retained. The porous nanofiber membrane was successfully prepared by mixed Wharton’s jelly with PCL. Live and dead staining and CCK-8 assay confirmed that the nanofiber membrane possessed perfect cytocompatibility. In vivo histological observation confirmed that the nanofiber membrane-chondrocytes construct could regenerate mature cartilage tissue. Conclusion The human umbilical cord Wharton’s jelly can be fabricated into a suitable scaffold by electrospinning technology for cartilage tissue engineering.