The exploration of alternative sources of extracellular matrix (ECM) is driven by increasing demand and the need for sustainable biomaterials. Tuna (Thunnus albacares) bones, a by-product of the fishing industry, represent a potential ECM source due to their abundance, collagen-rich composition, and biocompatibility. This study investigated the effects of demineralization using hydrochloric acid (HCl) at concentrations of 0.5 M and 1.0 M for varying durations (0.5, 1, 6, 12, and 24 h), and decellularization using sodium dodecyl sulfate (SDS) and Triton X-100 (TX100) at concentrations of 0.1%, 0.5%, and 1.0%. Demineralization effectively reduced inorganic content, with residual calcium levels dropping below 5% after 6 h. Kinetic analysis indicated a second-order reaction, and ATR–FTIR spectra confirmed the disappearance of phosphate and carbonate peaks alongside the preservation of amide bands. Demineralized bone matrix (dBM) retained good thermal stability and mechanical properties. Decellularization efficiency was assessed through H&E staining, dsDNA quantification, and SDS-PAGE analysis. Decellularized extracellular bone matrix (dEBM) treated with 1.0% SDS showed the lowest dsDNA levels (14.00 ± 7.94 ng/mg) and absence of cellular material. SDS-PAGE confirmed type I collagen preservation, particularly in samples treated with ≤0.5% SDS or TX100. This study establishes that tuna fish bones can be effectively processed into ECM, supporting their potential as a sustainable biomaterial for advanced biomedical applications.