Human G protein-coupled receptors (GPCRs), especially adrenoceptors, play a crucial role in maintaining important physiological activities including cardiovascular and pulmonary functions. Among all adrenoceptors, β-adrenoceptors are the best characterized GPCRs and possess distinctive features as drug targets. Similarly, ligands that activate/deactivate β-adrenoceptors also hold a significant position in the field of biomarker identification and drug discovery. Several studies regarding molecular characterization of the β-adrenoceptor ligands have revealed that ligands with abilities to inhibit basal or intrinsic receptor activity or prevent receptor desensitization are particularly important to efficiently manage detrimental health conditions, including chronic heart failure, asthma, chronic obstructive pulmonary disease, obesity, and diabetes. Given the importance of β-adrenoceptors as molecular targets for many pathological conditions, this review aims to provide a detailed insight on the structural and functional aspects of β-adrenoceptors, with a particular emphasis on their importance as biomarkers and therapeutic targets.