Signal amplification based on biofunctional nanomaterials has recently attracted considerable attention due to the need for ultrasensitive bioassays and the trend towards miniaturized assays. The biofunctional nanomaterials can not only produce a synergic effect among catalytic activity, conductivity and biocompatibility to accelerate the signal transduction, but also provide amplified recognition events by high loading of signal tags, leading to a highly sensitive and specific biosensing. Most importantly, nanoscaled materials are in direct contact with the environment, which permits them to act as chemical and biological sensors in single-molecule detection of biomolecules. In this tutorial review, we will focus on recent significant advances in signal amplification strategies combining the cross-disciplines of chemistry, biology, and materials science, and highlight some elegant applications of biofunctional nanomaterials as excellent electronic or optical signal tags in ultrasensitive bioanalysis. The biofunctional nanomaterials-based biosensing opens a series of concepts for basic research and offers new tools for detection of trace amounts of a wide variety of analytes in clinical, environmental, and industrial applications.