In this paper, two new flavonol derivatives, 2-(4-(dodecyloxy)phenyl)-3-hydroxyflavone (DHF) and 2-(3,5-bis-(dodecyloxy)phenyl)-3-hydroxyflavone (BDHF), were synthesized to investigate the respective self-assembly behaviors at the liquid/ solid interface by scanning tunneling microscopy. In addition, a linear pyridine derivative with acetylene groups called BisPy was added to regulate the assembly of DHF and BDHF, individually. However, only BDHF molecules successfully co-assembled into grid structures with BisPy molecules. Furthermore, the assembly and co-assembly behavior mechanism of flavonol derivatives and BisPy molecules were further studied by density functional theory calculations. This work will lay a foundation for investigating the self-assembly of flavonol derivatives and the co-assembly regulated by pyridine derivatives at the liquid−solid interface.