Decellularized bovine and porcine tissues have been used as scaffolds to support tissue regeneration but inherit religious restrictions and risks of disease transmission to humans. Decellularized marine tissues are seen as attractive alternatives due to their similarity to mammalian tissues, reduced biological risks, and less religious restrictions. The aim of this study was to derive an acellular scaffold from the skin of tilapia and evaluate its suitability as a tissue engineering scaffold. Tilapia skin was treated with a series of chemical and enzymatic treatments to remove cellular materials. The decellularized tilapia skin (DTS) was then characterized and evaluated in vitro and in vivo to assess its biological compatibility. The results indicated that the decellularization process removed 99.6% of the DNA content from tilapia skin. The resultant DTS was shown to possess a high denaturation temperature of 68.1 ± 1.0°C and a high Young's modulus of 56.2 ± 14.4 MPa. The properties of DTS were also compared against those of crosslinked electrospun tilapia collagen membrane, another form of tilapia-derived collagen scaffold. In vitro studies revealed that both DTS and crosslinked electrospun tilapia collagen promoted cellular metabolic activity, differentiation, and mineralization of murine preosteogenic MC3T3-E1 cells. The rat calvarial defect model was used to evaluate the in vivo performance of the scaffolds, and both scaffolds did not induce hyperacute rejections. Furthermore, they enhanced bone regeneration in the critical defect compared with the sham control. This study suggests that tilapia-derived scaffolds have great potential in tissue engineering applications.