Sample preparation is a crucial step for the reliable and accurate analysis of both small molecule and biopolymers which often involves processes such as isolation, pre-concentration, removal of interferences (purification), and pre-processing (e.g., enzymatic digestion) of targets from a complex matrix. Gold nanoparticle (GNP)-assisted sample preparation and pre-concentration has been extensively applied in many analytical procedures in recent years due to the favorable and unique properties of GNPs such as size-controlled synthesis, large surface-to-volume ratio, surface inertness, straightforward surface modification, easy separation requiring minimal manipulation of samples. This review article primarily focuses on applications of GNPs in sample preparation, in particular for bioaffinity capture and biocatalysis. In addition, their most common synthesis, surface modification and characterization methods are briefly summarized. Proper surface modification for GNPs designed in accordance to their target application directly influence their functionalities, e.g., extraction efficiencies, and catalytic efficiencies. Characterization of GNPs after synthesis and modification is worthwhile for monitoring and controlling the fabrication process to ensure proper quality and functionality. Parameters such as morphology, colloidal stability, and physical/chemical properties can be assessed by methods such as surface plasmon resonance, dynamic light scattering, zeta-potential determinations, transmission electron microscopy, Taylor dispersion analysis, and resonant mass measurement, among others. The accurate determination of the surface coverage appears to be also mandatory for the quality control of functionality of the nanoparticles. Some promising applications of (functionalized) GNPs for bioanalysis and sample preparation are described herein.