The hyperpolarization and cyclic nucleotide activated ion (HCN) channels have garnered increasing attention due to their association with the pathogenesis of various diseases. Given the heterogeneity in their expression patterns among different HCN channel subtypes, a comprehensive understanding of their function remains elusive and requires further investigation. The ion channel properties such as voltage dependence, are subject to modulation by endogenous cyclic nucleotides binding to the cyclic nucleotide binding domain (CNBD). In this study, we systematically assess the influence of seven distinct amino acids within the CNBD on ligand binding via the method of autodisplay and flow cytometry. Native HCN4 C-Linker-CNBD and its corresponding mutants were separately presented as fusion proteins on the surface of E. coli cells. Following incubation with 8-Fluo-cAMP, the whole cell fluorescence was quantified via flow cytometry. Perturbations in ligand binding, attributable to specific mutation, led to diminished fluorescence intensity (mFI) relative to the unaltered C-Linker-CNBD. Notably, mutations E660R, R669E and R710E resulted in a near-complete loss of whole cell fluorescence, confirming their importance for ligand binding as described before. Additionally, two amino acid substitutions, V642S and L652S, which were not anticipated to directly interact with the ligand, exhibited a strong reduction in mFI. We showed here for the first time the impact of these residues on ligand binding. Only moderate effects on ligand binding were observed for the mutants T670A and C662A. The presented ligand binding assay offers a rapid means of identifying residues essential for ligand binding. This information can be useful in the targeted drug design.