Cell cultured meat has been extensively studied as an environmentally friendly, energy-saving and more effective technology. However, there are many technical bottlenecks, especially the regulatory mechanism and manufacturing method of in vitro myogenesis. Based on an edible modified silk protein scaffold, with 3D culturing, in situ differentiated and transcriptome analysis, this study describes novel scaffolds and fabrication methods for cell cultured meat. The results showed that the effective space and utilization efficiency for cell culture of the scaffold is 26–1 000 that of the traditional culture dish; it could form a tissue-like structure. Transcriptomics revealed the regulatory pathways and key factors of different cycles. It clarifies that the multi-cycle process of myoblast myogenesis in vitro is different from the single feedback regulation in vivo. More importantly, a novel scaffold-based cell cultured meat manufacturing method was developed, further develop a new tissue culture solution that is different from existing cell culture meat production. For manufacturing processes, it provides a new cell culture meat technology system, provides a theoretical basis for the regulation of cell proliferation and muscle growth, and lays the technical foundation for in situ tissue culture of cell cultured meat in vitro.