The genetic information coded in DNA leads to trait innovation via a gene regulatory network (GRN) in development. Here, we developed a conserved non-coding element interpretation method to integrate multi-omics data into gene regulatory network (CNEReg) to investigate the ruminant multi-chambered stomach innovation. We generated paired expression and chromatin accessibility data during rumen and esophagus development in sheep and revealed 1601 active ruminant-specific conserved non-coding elements (active-RSCNEs). To interpret the function of these active-RSCNEs, we define toolkit transcription factors (TTFs) and model their regulation on rumen-specific genes via batteries of active-RSCNEs during development. Our developmental GRN revealed 18 TTFs and 313 active-RSCNEs regulating seven rumen functional modules. Notably, six TTFs (OTX1, SOX21, HOXC8, SOX2, TP63, and PPARG), as well as 16 active-RSCNEs, functionally distinguish the rumen from the esophagus. Our study provides a systematic approach to understanding how gene regulation evolves and shapes complex traits by putting evo-devo concepts into practice with developmental multi-omics data.