Gold nanoparticles (Au NPs) possess many advantages such as facile synthesis, controllable size and shape, good biocompatibility, and unique optical properties. Au NPs have been widely used in biomedical fields, such as hyperthermia, biocatalysis, imaging, and drug delivery. The broad application range may result in hazards to the environment and human health. Therefore, it is important to predict safety and evaluate therapeutic efficiency of Au NPs. It is necessary to establish proper approaches for the study of toxicity and biomedical effects. In this review, we first focus on the recent progress in biological effects of Au NPs at the molecular and cellular levels, and then introduce key techniques to study the interaction between Au NPs and proteins. Knowledge of the biomedical effects of Au NPs is significant for the rational design of functional nanomaterials and will help predict their safety and potential applications.