Nowadays, there is huge demand for biosensing in point-of-care and wearable formats. Label-free biosensors meet the criteria because they are fast and straightforward without the need for labeling to detect target molecules. Instead, they have to detect a small amount of analyte among irrelevant molecules with a high signal-to-noise ratio by performing efficient biorecognition. Therefore, it is crucial to design and control the structure of the selective layer at the interface between the sample solution and the transducer. The selective layer contains capturing elements from natural or synthetic origins. Artificial receptors are a new class of components with improved stability, durability, and quality as compared with natural antibodies and enzymes. This review addresses the functionalization of the selective layer. It possesses smart, biomimetic, and non-fouling characteristics using synthetic receptors and molecules. Molecular switches made of an oligonucleotide can respond to the target upon environmental stimuli. Their responsiveness is tunable by designing the probe sequence. Cell membranes in which bioreceptors are embedded in self-assembled phospholipid bilayer membranes provide hints for developing an advanced biosensing platform. Cell membrane-mimetic surfaces are robust through covalent contacts and resist nonspecific adsorption. The adaptation of a cell surface receptor efficiently improves the binding specificity. These technologies are essential for developing next-generation label-free biosensors.