Transcriptional regulation involves a series of sophisticated protein–protein and protein–DNA interactions (PPI and PDI). Some transcriptional complexes, such as c‐Fos/c‐Jun and their binding DNA fragments, have been conserved over the past one billion years. Considering the thermodynamic principle for transcriptional complex formation, we hypothesized that the c‐Fos/c‐Jun complex may represent a thermodynamic summit in the evolutionary space. To test this, we invented a new method, termed One‐Pot‐seq, which combines cDNA display and proximity ligation to analyse PPI/PDI complexes simultaneously. We found that the wild‐type c‐Fos/c‐Jun complex is indeed the most thermodynamically stable relative to various mutants of c‐Fos/c‐Jun and binding DNA fragments. Our method also provides a universal approach to detect transcriptional complexes and explore transcriptional regulation mechanisms. [ABSTRACT FROM AUTHOR]