Mineralized　collagen　is　the　basic　unit　in　hierarchically　organized　natural　bone　with　different　structures.　Polyacrylic　acid　(PAA)　and　periodic　fluid　shear　stress　(FSS)　are　the　most　common　chemical　and　physical　means　to　induce　intrafibrillar　mineralization.　In　the　present　study,　non-mineralized　collagen,　extrafibrillar　mineralized　(EM)　collagen,　intrafibrillar　mineralized　(IM)　collagen,　and　hierarchical　intrafibrillar　mineralized　(HIM)　collagen　induced　by　PAA　and　FSS　were　prepared,　respectively.　The　physical　and　chemical　properties　of　these　mineralized　collagens　with　different　microstructures　were　systematically　investigated　afterwards.　Transmission　electron　microscopy　(TEM)　and　scanning　electron　microscopy　(SEM)　showed　that　mineralized　collagen　with　different　microstructures　was　prepared　successfully.　The　pore　density　of　the　mineralized　collagen　scaffold　is　higher　under　the　action　of　periodic　FSS.　Fourier　transform　infrared　spectroscopy　(FTIR)　analysis　showed　the　formation　of　the　hydroxyapatite　(HA)　crystal.　A　significant　improvement　in　the　pore　density,　hydrophilicity,　enzymatic　stability,　and　thermal　stability　of　the　mineralized　collagen　indicated　that　the　IM　collagen　under　the　action　of　periodic　FSS　was　beneficial　for　maintaining　collagen　activity.　HIM　collagen　fibers,　which　are　prepared　under　the　co-action　of　periodic　FSS　and　sodium　tripolyphosphate　(TPP),　may　pave　the　way　for　new　bone　substitute　material　applications.