Man-made materials are easily available, processed and modified during bone defect repairing, without worrying about potential infections, immunological incompatibility, sterility, or donor site morbidity. Collagen (Col)/nano-hydroxyapatite (nHA) composite nanofibers based on ordered nHA ceramic fibers was prepared by sintering electrostatic spinning PVP/HA composite fibers followed by impregnation with collagen solution. Without requiring strict control of temperature or humidity, this method prevented nHA aggregation during the sintering process and reduced the use of costly Col as compared with direct electrospinning. The composite nanofiber membrane with bone-like components and structures has a great potential for application in bone tissue engineering. And the method implement easily.. The mineralization and degradation characteristics of the nanofiber biocomposite are beneficial for the bone conductivity and MSCs growth and metabolism. Protein adsorption of Col/HA nanofiber biocomposite is outstanding. There is no significant difference in cells viability between the experiment group and the control group, and Col/HA nanofibers biocomposite has weak cytotoxicity to MSCs. Overall, the approach successfully prepared Col/HA nanofibers biocomposite at common electrospinning environment with uniformly distributed high content nHA, low Col concentration and less Col consumption, all of which make it an excellent choice for the bone tissue engineering.