For the wide application of nanoparticles (NPs) (e.g., in nanotribology), it is of fundamental and practical importance to understand the self-assembly and lubrication behavior of confined NPs. In this work, a systematic study was conducted to probe the assembly and associated surface forces of spherical gold nanoparticles (Au NPs, diameter ∼5 nm) confined between pairs of mica (negatively charged) and (3-aminopropyl)triethoxysilane modified mica (APTES-mica, positively charged) surfaces using a surface forces apparatus (SFA) under aqueous conditions. It is observed that Au NPs were squeezed out of the confined gap between two mica surfaces during the loading process, resulting from the repulsive electric-double layer force. In contrast, multilayers of Au NPs were confined between two APTES-mica surfaces because of the attractive double-layer force between oppositely charged Au NPs and APTES-mica. Interestingly, the interaction between Au NPs and APTES-mica is stronger than the interactions between Au NPs, resulting in the rearrangement of the confined Au NPs under shearing. Importantly, a large friction coefficient (μ > 0.7) with unexpected nonlinear stick-slip friction was observed when sliding two APTES-mica surfaces with thin layers of Au NPs (∼20 nm) confined in between. The observed stick-slip motion could be explained by the velocity-dependent friction model where a critical shear velocity was required for transiting from stick-slip to smooth sliding. Our study provides useful information on the assembly and interaction forces of confined nanoparticles on charged surfaces, with implications for predicting the behaviors of NPs under confinement in various engineering applications.