Here, we aim at developing a novel biomatrix from decellularized bovine spinal meninges for tissue engineering and regenerative medicine applications. Within this concept, the bovine spinal meninges were decellularized using 1% Triton X-100 for 48 h, and residual nuclear content was determined with double-strand DNA content analysis and agarose gel electrophoresis. The major matrix components such as sulfated GAGs and collagen before and after the decellularization process were analyzed with DMMB, hydroxyproline assay and SDS-PAGE. Subsequently, the native bovine spinal meninges (nBSM) and decellularized BSM (dBSM) were physiochemically characterized via ATR-FTIR spectroscopy, TGA, DMA and tensile strength test. The dsDNA content in the nBSM was 153.39 +/- 53.93 ng/mg dry weight, versus in the dBSM was 39.47 +/- 4.93 ng/mg (n = 3) dry weight and DNA fragments of more than 200 bp in length were not detected in the dBSM by agarose gel electrophoresis. The sulfated GAGs contents for nBSM and dBSM were observed to be 10.87 +/- 1.2 and 11.42 +/- 2.01 mu g/mg dry weight, respectively. The maximum strength of dBSM in dry and wet conditions was found to be 19.67 +/- 0.21 MPa and 13.97 +/- 0.17 MPa, while nBSM (dry) was found to be 26.26 +/- 0.28 MPa. MTT, SEM, and histology results exhibited that the cells attached to the surface of dBSM, and proliferated on the dBSM. In conclusion, the in vitro preliminary study has demonstrated that the dBSM might be a proper and new bioscaffold for tissue engineering and regenerative medicine applications.