Abstract: Hdroxyapatite(HA) was prepared using porcine trabecular bone after being degreased, deproteined, calcined and ball milling. HA/chitosan(CS) composite membranes were prepared by blending and drying HA and CS solution. The effect of milling conditions on the HA particle size was inspected by the orthogonal design. MC3T3-E1 preosteoblasts were seeded on the surface of the composite membranes, and then cell morphology and proliferation were detected by SEM and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT) methods. The results show that the prepared HA is relatively homogeneous micrometer-scale spherical particle with median diameter(D₅₀) in 1.21~1.67 μm range, and HA particles are distributed uniformly in the matrix, HA particle and CS combined closely. Composite membrane has good mechanical properties, MC3T3-E1 cells can adhere and grow well on the surface of composite membrane. The cell proliferation results show that composite membrane under the preparation conditions: calcination temperature 1000 ℃, the milling ball propotion 4∶4∶2, milling rate 230 r·min^-1, milling time 2.5 h, HA∶CS =5∶5 (mass ratio) is the most beneficial for cell proliferation.