<jats:title>Abstract</jats:title><jats:p>Extracellular matrix (<jats:styled-content style="fixed-case">ECM</jats:styled-content>) hydrogels are used as scaffolds to facilitate the repair and reconstruction of tissues. This study aimed to optimize the decellularization process of porcine skeletal muscle <jats:styled-content style="fixed-case">ECM</jats:styled-content> and to formulate a matrix hydrogel scaffold. Five multi‐step methods (methods A–E) were used to generate acellular <jats:styled-content style="fixed-case">ECM</jats:styled-content> from porcine skeletal muscle [rinsing in <jats:styled-content style="fixed-case">SDS</jats:styled-content>, trypsin, ethylenediaminetetraacetic acid (<jats:styled-content style="fixed-case">EDTA</jats:styled-content>), Triton X‐100 and/or sodium deoxycholate at 4–37°C]. The resulting <jats:styled-content style="fixed-case">ECM</jats:styled-content> was evaluated using haematoxylin and eosin, 4‐6‐diamidino‐2‐phenylindole (DAPI) staining, and <jats:styled-content style="fixed-case">DNA</jats:styled-content> quantification. Acellular matrix was dissolved in pepsin and gelled at 37°C. Hydrogel response to temperature was observed <jats:italic>in vivo</jats:italic> and <jats:italic>in vitro</jats:italic>. <jats:styled-content style="fixed-case">ECM</jats:styled-content> components were assessed by Masson, Sirius red, and alcian blue staining, and total protein content. Acellular porcine skeletal muscle exhibited a uniform translucent white appearance. No intact nuclear residue was detected by haematoxylin and eosin staining, while <jats:styled-content style="fixed-case">DAPI</jats:styled-content> staining showed a few nuclei in the matrixes produced by methods B, C, and D. Method A generated a gel that was too thin for gelation. However, the matrix obtained by rinsing in 0.2% trypsin/0.1% <jats:styled-content style="fixed-case">EDTA</jats:styled-content>, 0.5% Triton X‐100, and 1% Triton X‐100/0.2% sodium deoxycholate was nuclei‐free and produced a viscous solution that formed a structurally stable white jelly‐like hydrogel. The residual <jats:styled-content style="fixed-case">DNA</jats:styled-content> content of this solution was 49.37 ± 0.72 ng/mg, significantly less than in fresh skeletal muscle, and decreased to 19.22 ± 0.85 ng/mg after gelation (<jats:italic>P</jats:italic> < 0.05). The acellular matrix was rich in collagen and glycosaminoglycan, with a total protein concentration of 64.8 ± 6.9%. An acellular <jats:styled-content style="fixed-case">ECM</jats:styled-content> hydrogel from porcine skeletal muscle was efficiently produced.</jats:p>