Polyampholytes are unique macromolecules containing acid/base or anionic/cationic groups in the main or side chains that mimic the behavior of proteins, polypeptides or polynucleotides. Water-soluble and water-swelling polyampholytes exhibit salt-tolerant, thermal-resistant, shear-stable, self-healing, anti-fouling, self-assembling, and stimuli-responsive properties that provide broad impact as structural biomaterials, drug delivery and chemo-mechanical systems, biosensors, energy storage devices, supercapacitors, actuators etc. In this review we mainly pay attention to fundamental and applied aspects of synthetic polyampholytes in the context of biotechnology and medicine. Among the outlined topics the role of linear and crosslinked amphoteric macromolecules as well as macromolecular complexes of polyampholytes with polyelectrolytes, DNA, proteins and drugs is highlighted. Cryoprotective behavior of synthetic polyampholytes is shown with respect to cell preservation and viability. Injectable, soft, flexible, stretchable, self-healing and stimuli-responsive physically or chemically crosslinked polyampholyte gels that are promising materials for fabrication of artificial tissue, wound dressing, cartilage, cardiovascular, bone defect repair, regenerative medicine and tissue engineering scaffolds, soft actuators and sensors, soft robotics are also in the eyeshot of this review. Hierarchically structured multi-responsive polyampholytic gels, multilayered films and membranes possessing encapsulation–release and “on–off” behaviors are considered as antibiofouling, anticoagulation, and drug delivery system.