We report the application of bioChIP-seq, bulk RNA-seq, Hi-C, H3K27ac HiChIP, and Massively parallel reporter assays (MPRAs) to characterize the p300-bound regulatory regions in murine cardiomyocytes (CMs). By obtaining ChIP-seq data of coactivator p300 from seven developmental stages of mouse CMs, we defined the dynamic p300 enhancers from embryonic CMs to adult CMs. We then validated the activity of dynamic p300 enhancers with AAV9-based MPRAs, we found dynamic p300 enhancers show dynamic activity from postnatal day 0 (P0) CMs to 4-week-old CMs. In addition, MRPA results suggest nuclear receptor motifs are required for the activity of some p300 late enhancers. With Hi-C and H3K27ac HiChIP data of E12.5, P0 and adult CMs, we identified chromatin structure changes such as chromatin loops, compartment switch, and new TAD boundaries are associated with dynamic p300 binding. This study provides data source of CM-selective p300 enhancers, chromatin 3D structure and gene expression during CM development and maturation. Overall design: Identification and characterization of p300 enhancers by CM-selective p300 bioChIP-seq, MPRAs, RNA-sq, Hi-C, and H3K27ac HiChIP. The processed data files for GSE222160 were updated on March 10, 2023.