The current study presents porous Ti-40Nb alloys fabricated by one step spark plasma sintering (SPS) technique with space holder (NH4HCO3) method. The microstructures and mechanical properties of the porous Ti-40Nb alloys with a different volume ratio of space holder were investigated byXRD analysis, scanning electron microscope (SEM) and compression tests. Rat bone marrow mesenchymal stem cells (rBMSCs) were used to assess the in vitro biocompatibility of porous Ti-40Nb alloys. Results showed that porous Ti-40Nb alloys consisted of β and αphase. The pore characteristics of the Ti- 40Nb alloy could be controlled by the combination process of SPS technique and space holder method. With different volume ratios (10%–50%) of the space-holder (NH4HCO3) added, Ti-40Nb alloys possessed interconnected structures with micro/macro-pores and 13.4%–61.2% porosity. The elastic modulus and compression strength of porous Ti-40Nb alloys were in the range of 18.4–27.3 GPa/326–452 MPa, which were close to the natural human bone. Cell morphology and cell proliferation studies showed good cell adherence and growth on all group of alloys, suggesting nontoxicity and great biocompatibility of Ti-40Nb alloys. Furthermore, increasing the porosity of porous Ti-40Nb alloys cloud promoted the biological performance. Our work indicates that SPS could fabricate porous Ti-40Nb alloys with interconnected porous structures, such type Ti alloy biomaterials hold great potentials for future medical applications.