The hydroxyapatite/collagen (HAp/Col) nanocomposite membrane was prepared from the HAp/Col nanocomposite fibers. The HAp/Col membrane crosslinked by a vacuum heating showed 14±2 (dry) and 1.0±0.1 (wet) MPa in tensile strengths that were enough strength to operate in both cell culture and surgical operations. Histological observation and total DNA amount analysis demonstrated that human osteoblastic cells, MG63, derived from osteosarcoma on the HAp/Col membrane adhered and proliferated well as the same as those on tissue culture polystyrene (TCPS). Real-time polymerase chain reaction analyses indicated that alkaline phosphatase gene expression on the HAp/Col membrane was 1.4 times greater than that on TCPS. These results suggested that the HAp/Col nanocomposite membrane can be utilized in bone tissue engineering scaffold as well as bone filler.